Pyrrolobenzodiazepine-antibody conjugates

ABSTRACT

The present invention relates to pyrrolobenzodiazepines (PBDs) having a labile protecting group in the form of a linker to an antibody.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national phase application under 35 U.S.C. § 371of PCT International Application No. PCT/EP2018/053188, filed Feb. 8,2018, which claims the benefit of Great Britain Application No.1702031.4, filed 8 Feb. 8 2017, Great Britain Application No. 1719391.3,filed Nov. 22, 2017, Great Britain Application No. 1719398.8, filed Nov.22, 2017, and Great Britain Application No. 1719393.9, filed Nov. 22,2017, each of which is herein incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to pyrrolobenzodiazepines (PBDs) having alabile protecting group in the form of a linker to an antibody.

BACKGROUND TO THE INVENTION

Pyrrolobenzodiazepines

Some pyrrolobenzodiazepines (PBDs) have the ability to recognise andbond to specific sequences of DNA; the preferred sequence is PuGPu. Thefirst PBD antitumour antibiotic, anthramycin, was discovered in 1965(Leimgruber, et al., J. Am. Chem. Soc., 87, 5793-5795 (1965);Leimgruber, et al., J. Am. Chem. Soc., 87, 5791-5793 (1965)). Sincethen, a number of naturally occurring PBDs have been reported, and over10 synthetic routes have been developed to a variety of analogues(Thurston, et al., Chem. Rev. 1994, 433-465 (1994); Antonow, D. andThurston, D. E., Chem. Rev. 2011 111 (4), 2815-2864). Family membersinclude abbeymycin (Hochlowski, et al., J. Antibiotics, 40, 145-148(1987)), chicamycin (Konishi, et al., J. Antibiotics, 37, 200-206(1984)), DC-81 (Japanese Patent 58-180 487; Thurston, et al., Chem.Brit., 26, 767-772 (1990); Bose, et al., Tetrahedron, 48, 751-758(1992)), mazethramycin (Kuminoto, et al., J. Antibiotics, 33, 665-667(1980)), neothramycins A and B (Takeuchi, et al., J. Antibiodcs, 29,93-96 (1976)), porothramycin (Tsunakawa, et al., J. Antibiotics, 41,1366-1373 (1988)), prothracarcin (Shimizu, et al, J. Antibiotics, 29,2492-2503 (1982); Langley and Thurston, J. Org. Chem., 52, 91-97(1987)), sibanomicin (DC-102)(Hara, et al., J. Antibiotics, 41, 702-704(1988); Itoh, et al., J. Antibiotics, 41, 1281-1284 (1988)), sibiromycin(Leber, et al., J. Am. Chem. Soc., 110, 2992-2993 (1988)) and tomamycin(Arima, et al., J. Antibiotics, 25, 437-444 (1972)). PBDs are of thegeneral structure:

They differ in the number, type and position of substituents, in boththeir aromatic A rings and pyrrolo C rings, and in the degree ofsaturation of the C ring. In the B-ring there is either an imine (N═C),a carbinolamine (NH—CH(OH)), or a carbinolamine methyl ether(NH—CH(OMe)) at the N10-C11 position which is the electrophilic centreresponsible for alkylating DNA. All of the known natural products havean (S)-configuration at the chiral C11a position which provides themwith a right-handed twist when viewed from the C ring towards the Aring. This gives them the appropriate three-dimensional shape forisohelicity with the minor groove of B-form DNA, leading to a snug fitat the binding site (Kohn, In Antibiotics III. Springer-Veriag, NewYork, pp. 3-11 (1975); Hurley and Needham-VanDevanter, Acc. Chem. Res.,19, 230-237 (1986)). Their ability to form an adduct in the minorgroove, enables them to interfere with DNA processing, hence their useas antitumour agents.

One pyrrolobenzodiazepine compound is described by Gregson et al. (Chem.Commun. 1999, 797-798) as compound 1, and by Gregson et al. (J. Med.Chem. 2001, 44, 1161-1174) as compound 4a. This compound, also known asSG2000, is shown below:

WO 2007/085930 describes the preparation of dimer PBD compounds havinglinker groups for connection to a cell binding agent, such as anantibody. The linker is present in the bridge linking the monomer PBDunits of the dimer.

Dimer PBD compounds having linker groups for connection to a cellbinding agent, such as an antibody, have been described in WO2011/130613 and WO 2011/130616. The linker in these compounds isattached to the PBD core via the C2 position, and are generally cleavedby action of an enzyme on the linker group. In WO 2011/130598, thelinker in these compounds is attached to one of the available N10positions on the PBD core, and are generally cleaved by action of anenzyme on the linker group.

Antibody-Drug Conjugates

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders (Carter, P.(2006) Nature Reviews Immunology 6:343-357). The use of antibody-drugconjugates (ADC), i.e. immunoconjugates, for the local delivery ofcytotoxic or cytostatic agents, i.e. drugs to kill or inhibit tumorcells in the treatment of cancer, targets delivery of the drug moiety totumors, and intracellular accumulation therein, whereas systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells (Xie et al (2006)Expert. Opin. Biol. Ther. 6(3):281-291; Kovtun et al (2006) Cancer Res.66(6):3214-3121; Law et al (2006) Cancer Res. 66(4):2328-2337; Wu et al(2005) Nature Biotech. 23(9):1137-1145; Lambert J. (2005) Current Opin.in Pharmacol. 5:543-549; Hamann P. (2005) Expert Opin. Ther. Patents15(9):1087-1103; Payne, G. (2003) Cancer Cell 3:207-212; Trail et al(2003) Cancer Immunol. Immunother. 52:328-337; Syrigos and Epenetos(1999) Anticancer Research 19:605-614).

Maximal efficacy with minimal toxicity is sought thereby. Efforts todesign and refine ADC have focused on the selectivity of monoclonalantibodies (mAbs) as well as drug mechanism of action, drug-linking,drug/antibody ratio (loading), and drug-releasing properties (Junutula,et al., 2008b Nature Biotech., 26(8):925-932; Doman et al (2009) Blood114(13):2721-2729; U.S. Pat. Nos. 7,521,541; 7,723,485; WO2009/052249;McDonagh (2006) Protein Eng. Design & Sel. 19(7): 299-307; Doronina etal (2006) Bioconj. Chem. 17:114-124; Erickson et al (2006) Cancer Res.66(8):1-8; Sanderson et al (2005) Clin. Cancer Res. 11:843-852; Jeffreyet al (2005) J. Med. Chem. 48:1344-1358; Hamblett et al (2004) Clin.Cancer Res. 10:7063-7070). Drug moieties may impart their cytotoxic andcytostatic effects by mechanisms including tubulin binding, DNA binding,proteasome and/or topoisomerase inhibition. Some cytotoxic drugs tend tobe inactive or less active when conjugated to large antibodies orprotein receptor ligands.

The present inventors have developed particular PBD dimer antibodyconjugates.

SUMMARY OF THE INVENTION

A first aspect of the present invention provides a conjugate of formula(I):Ab-(DL)_(p)  (I)

wherein:

Ab is an antibody that binds to DLK1;

DL is

wherein:

X is selected from the group comprising: a single bond, —CH₂— and—C₂H₄—;

n is from 1 to 8;

m is 0 or 1;

R⁷ is either methyl or phenyl;

when there is a double bond between C2 and C3, R² is selected the groupconsisting of:

(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁-5 saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2 and C3, R² is

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

when there is a double bond between C2′ and C3′, R¹² is selected thegroup consisting of:

(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁₋₅ saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(35a) and R^(35b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2′ and C3′, R¹² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

and p is from 1 to 8.

A second aspect of the present invention provides a conjugate of formula(I):Ab-(DL)p  (I)

wherein:

Ab is an antibody that binds to KAAG1; and

DL is as defined for the first aspect, above.

A third aspect of the present invention provides a conjugate of formula(I):Ab-(DL)p  (I)

wherein:

Ab is an antibody that binds to Mesothelin; and

DL is as defined for the first aspect, above.

These conjugates have been found to exhibit good activity, andsurprising tolerability compared to analogous conjugates not containingthe sulfonamido moiety.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the in vitro cytotoxicity of a conjugate according to thefirst aspect of the invention;

FIG. 2 shows the in vivo efficacy of a conjugate according to the firstaspect of the invention.

FIG. 3 shows in vitro cytotoxicity in A204 and Hep3B cells in 2D and 3Dcell culture of a conjugate according to the first aspect of theinvention.

FIG. 4 shows the in vitro cytotoxicity of a conjugate according to thesecond aspect of the invention.

FIG. 5 shows the in vivo efficacy of a conjugate according to the secondaspect of the invention.

FIG. 6 shows the in vivo anti-tumor activity in SN12C xenograft model ofa conjugate according to the second aspect of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a PBD dimer with a linker connectedthrough the N10 position on one of the PBD moieties conjugated to anantibody as defined below.

The present invention is suitable for use in providing a PBD compound toa preferred site in a subject. The conjugate allows the release of anactive PBD compound that does not retain any part of the linker. Thereis no stub present that could affect the reactivity of the PBD compound.Thus the conjugate of formula (I) would release the compound Relax:

The specified link between the PBD dimer and the antibody in the presentinvention is preferably stable extracellularly. Before transport ordelivery into a cell, the antibody-drug conjugate (ADC) is preferablystable and remains intact, i.e. the antibody remains linked to the drugmoiety. The linkers are stable outside the target cell and may becleaved at some efficacious rate inside the cell. An effective linkerwill: (i) maintain the specific binding properties of the antibody; (ii)allow intracellular delivery of the conjugate or drug moiety; (iii)remain stable and intact, i.e. not cleaved, until the conjugate has beendelivered or transported to its targeted site; and (iv) maintain acytotoxic, cell-killing effect or a cytostatic effect of the PBD drugmoiety. Stability of the ADC may be measured by standard analyticaltechniques such as mass spectroscopy, HPLC, and the separation/analysistechnique LC/MS.

Delivery of the compounds of formulae RelA is achieved at the desiredactivation site of the conjugate of formula (I) by the action of anenzyme, such as cathepsin, on the linking group, and in particular onthe valine-alanine dipeptide moiety.

Definition

Substituents

The phrase “optionally substituted” as used herein, pertains to a parentgroup which may be unsubstituted or which may be substituted.

Unless otherwise specified, the term “substituted” as used herein,pertains to a parent group which bears one or more substituents. Theterm “substituent” is used herein in the conventional sense and refersto a chemical moiety which is covalently attached to, or if appropriate,fused to, a parent group. A wide variety of substituents are well known,and methods for their formation and introduction into a variety ofparent groups are also well known.

Examples of substituents are described in more detail below.

C₁₋₁₂ alkyl: The term “C₁₋₁₂ alkyl” as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from a carbonatom of a hydrocarbon compound having from 1 to 12 carbon atoms, whichmay be aliphatic or alicyclic, and which may be saturated or unsaturated(e.g. partially unsaturated, fully unsaturated). The term “C₁₋₄ alkyl”as used herein, pertains to a monovalent moiety obtained by removing ahydrogen atom from a carbon atom of a hydrocarbon compound having from 1to 4 carbon atoms, which may be aliphatic or alicyclic, and which may besaturated or unsaturated (e.g. partially unsaturated, fullyunsaturated). Thus, the term “alkyl” includes the sub-classes alkenyl,alkynyl, cycloalkyl, etc., discussed below.

Examples of saturated alkyl groups include, but are not limited to,methyl (C₁), ethyl (C₂), propyl (C₃), butyl (C₄), pentyl (C₅), hexyl(C₆) and heptyl (C₇).

Examples of saturated linear alkyl groups include, but are not limitedto, methyl (C₁), ethyl (C₂), n-propyl (C₃), n-butyl (C₄), n-pentyl(amyl) (C₅), n-hexyl (C₆) and n-heptyl (C₇).

Examples of saturated branched alkyl groups include iso-propyl (C₃),iso-butyl (C₄), sec-butyl (C₄), tert-butyl (C₄), iso-pentyl (C₅), andneo-pentyl (C₅).

C₂₋₁₂ Alkenyl: The term “C₂₋₁₂ alkenyl” as used herein, pertains to analkyl group having one or more carbon-carbon double bonds.

Examples of unsaturated alkenyl groups include, but are not limited to,ethenyl (vinyl, —CH═CH₂), 1-propenyl (—CH═CH—CH₃), 2-propenyl (allyl,—CH—CH═CH₂), isopropenyl (1-methylvinyl, —C(CH₃)═CH₂), butenyl (C₄),pentenyl (C5), and hexenyl (C₆).

C₂₋₁₂ alkynyl: The term “C₂₋₁₂ alkynyl” as used herein, pertains to analkyl group having one or more carbon-carbon triple bonds.

Examples of unsaturated alkynyl groups include, but are not limited to,ethynyl (—C≡CH) and 2-propynyl (propargyl, —CH₂—C≡CH).

C₃₋₁₂ cycloalkyl: The term “C₃₋₁₂ cycloalkyl” as used herein, pertainsto an alkyl group which is also a cyclyl group; that is, a monovalentmoiety obtained by removing a hydrogen atom from an alicyclic ring atomof a cyclic hydrocarbon (carbocyclic) compound, which moiety has from 3to 7 carbon atoms, including from 3 to 7 ring atoms.

Examples of cycloalkyl groups include, but are not limited to, thosederived from:

saturated monocyclic hydrocarbon compounds:

cyclopropane C₃), cyclobutane C₄), cyclopentane (C5), cyclohexane (C6),cycloheptane (C₇), methylcyclopropene (C₄), dimethylcyclopropane (C₅),methylcyclobutane (C₅), dimethylcyclobutane (C₆), methylcyclopentane(C₆), dimethylcyclopentane (C₇) and methylcyclohexane (C₇);

unsaturated monocyclic hydrocarbon compounds:

cyclopropene C₃), cyclobutene C₄), cyclopentene (C5), cyclohexene (C6),methylcyclopropene (C₄), dimethylcyclopropene (C₅), methylcyclobutene(C₅), dimethylcyclobutene (C₆), methylcyclopentene (C₆),dimethylcyclopentene (C₇) and methylcyclohexene (C₇); and

saturated polycyclic hydrocarbon compounds:

norcarane C₇), norpinane C₇), norbornane C₇).

C₃₋₂₀ heterocyclyl: The term “C₃₋₂₀ heterocyclyl” as used herein,pertains to a monovalent moiety obtained by removing a hydrogen atomfrom a ring atom of a heterocyclic compound, which moiety has from 3 to20 ring atoms, of which from 1 to 10 are ring heteroatoms. Preferably,each ring has from 3 to 7 ring atoms, of which from 1 to 4 are ringheteroatoms.

In this context, the prefixes (e.g. C3-20, C3-7, 05-6, etc.) denote thenumber of ring atoms, or range of number of ring atoms, whether carbonatoms or heteroatoms. For example, the term “C₅₋₆ heterocyclyl”, as usedherein, pertains to a heterocyclyl group having 5 or 6 ring atoms.

Examples of monocyclic heterocyclyl groups include, but are not limitedto, those derived from:

N₁: aziridine (C₃), azetidine (C₄), pyrrolidine (tetrahydropyrrole)(C₅), pyrroline (e.g., 3-pyrroline, 2,5-dihydropyrrole) (C₅), 2H-pyrroleor 3H-pyrrole (isopyrrole, isoazole) (C₅), piperidine (C₆),dihydropyridine (C₆), tetrahydropyridine (C₆), azepine (C₇);

O₁: oxirane (C₃), oxetane (C₄), oxolane (tetrahydrofuran) (C₅), oxole(dihydrofuran) (C₅), oxane (tetrahydropyran) (C₆), dihydropyran (C₆),pyran (C₆), oxepin (C₇);

S₁: thiirane (C₃), thietane (C₄), thiolane (tetrahydrothiophene) (C₅),thiane (tetrahydrothiopyran) (C₆), thiepane (C₇);

O₂: dioxolane (C₅), dioxane (C₆), and dioxepane (C₇);

O₃: trioxane (C6);

N₂: imidazolidine (C₅), pyrazolidine (diazolidine) (C₅), imidazoline(C5), pyrazoline (dihydropyrazole) (C₅), piperazine (C₆);

N₁O₁: tetrahydrooxazole (C₅), dihydrooxazole (C₅), tetrahydroisoxazole(C₅), dihydroisoxazole (C₅), morpholine (C₆), tetrahydrooxazine (C₆),dihydrooxazine (C₆), oxazine (C₆);

N₁S₁: thiazoline (C₅), thiazolidine (C₅), thiomorpholine (C₆);

N₂O₁: oxadiazine (C₆);

O₁S₁: oxathiole (C₅) and oxathiane (thioxane) (C₆); and,

N₁O₁S₁: oxathiazine (C₆).

Examples of substituted monocyclic heterocyclyl groups include thosederived from saccharides, in cyclic form, for example, furanoses (C₅),such as arabinofuranose, lyxofuranose, ribofuranose, and xylofuranse,and pyranoses (C₆), such as allopyranose, altropyranose, glucopyranose,mannopyranose, gulopyranose, idopyranose, galactopyranose, andtalopyranose.

C₅₋₂₀ aryl: The term “C₅₋₂₀ aryl”, as used herein, pertains to amonovalent moiety obtained by removing a hydrogen atom from an aromaticring atom of an aromatic compound, which moiety has from 3 to 20 ringatoms. The term “C₅₋₇ aryl”, as used herein, pertains to a monovalentmoiety obtained by removing a hydrogen atom from an aromatic ring atomof an aromatic compound, which moiety has from 5 to 7 ring atoms and theterm “C₅₋₁₀ aryl”, as used herein, pertains to a monovalent moietyobtained by removing a hydrogen atom from an aromatic ring atom of anaromatic compound, which moiety has from 5 to 10 ring atoms. Preferably,each ring has from 5 to 7 ring atoms.

In this context, the prefixes (e.g. C₃₋₂₀, C₅₋₇, C₅₋₆, C₅₋₁₀, etc.)denote the number of ring atoms, or range of number of ring atoms,whether carbon atoms or heteroatoms. For example, the term “C₅₋₆ aryl”as used herein, pertains to an aryl group having 5 or 6 ring atoms.

The ring atoms may be all carbon atoms, as in “carboaryl groups”.

Examples of carboaryl groups include, but are not limited to, thosederived from benzene (i.e. phenyl) (C₆), naphthalene (C₁₀), azulene(C₁₀), anthracene (C₁₄), phenanthrene (C₁₄), naphthacene (C₁₈), andpyrene (C₁₆).

Examples of aryl groups which comprise fused rings, at least one ofwhich is an aromatic ring, include, but are not limited to, groupsderived from indane (e.g. 2,3-dihydro-1H-indene) (C₉), indene (C₉),isoindene (C₉), tetraline (1,2,3,4-tetrahydronaphthalene (C₁₀),acenaphthene (C₁₂), fluorene (C₁₃), phenalene (C₁₃), acephenanthrene(C₁₅), and aceanthrene (C₁₆).

Alternatively, the ring atoms may include one or more heteroatoms, as in“heteroaryl groups”. Examples of monocyclic heteroaryl groups include,but are not limited to, those derived from:

N₁: pyrrole (azole) (C₅), pyridine (azine) (C₅);

O₁: furan (oxole) (C₅);

S₁: thiophene (thiole) (C₅);

N₁O₁: oxazole (C₅), isoxazole (C₅), isoxazine (C₆);

N₂O₁: oxadiazole (furazan) (C₅);

N₃O₁: oxatriazole (C₅);

N₁S₁: thiazole (C₅), isothiazole (C₅);

N₂: imidazole (1,3-diazole) (C₅), pyrazole (1,2-diazole) (C₅),pyridazine (1,2-diazine) (C₅), pyrimidine (1,3-diazine) (C₅) (e.g.,cytosine, thymine, uracil), pyrazine (1,4-diazine) (C₆);

N₃: triazole (C₅), triazine (C₅); and,

N₄: tetrazole (C₅).

Examples of heteroaryl which comprise fused rings, include, but are notlimited to:

-   -   C₉ (with 2 fused rings) derived from benzofuran (O₁),        isobenzofuran (O₁), indole (N₁), isoindole (N₁), indolizine        (N₁), indoline (N₁), isoindoline (N₁), purine (N₄) (e.g.,        adenine, guanine), benzimidazole (N₂), indazole (N₂),        benzoxazole (N₁O₁), benzisoxazole (N₁O₁), benzodioxole (O₂),        benzofurazan (N₂O₁), benzotriazole (N₃), benzothiofuran (S₁),        benzothiazole (N₁S₁), benzothiadiazole (N₂S);    -   C₁₀ (with 2 fused rings) derived from chromene (O₁), isochromene        (O₁), chroman (O₁), isochroman (O₁), benzodioxan (O₂), quinoline        (N₁), isoquinoline (N₁), quinolizine (N₁), benzoxazine (N₁O₁),        benzodiazine (N₂), pyridopyridine (N₂), quinoxaline (N₂),        quinazoline (N₂), cinnoline (N₂), phthalazine (N₂),        naphthyridine (N₂), pteridine (N₄);    -   C11 (with 2 fused rings) derived from benzodiazepine (N₂);    -   C₁₃ (with 3 fused rings) derived from carbazole (N₁),        dibenzofuran (O₁), dibenzothiophene (S₁), carboline (N₂),        perimidine (N₂), pyridoindole (N₂); and,

C₁₄ (with 3 fused rings) derived from acridine (N₁), xanthene (O₁),thioxanthene (S₁), oxanthrene (O₂), phenoxathiin (O₁S₁), phenazine (N₂),phenoxazine (N₁O₁), phenothiazine (N₁S₁), thianthrene (S₂),phenanthridine (N₁), phenanthroline (N₂), phenazine (N₂).

The above groups, whether alone or part of another substituent, maythemselves optionally be substituted with one or more groups selectedfrom themselves and the additional substituents listed below.

Halo: —F, —Cl, —Br, and —I.

Hydroxy: —OH.

Ether: —OR, wherein R is an ether substituent, for example, a C₁₋₇ alkylgroup (also referred to as a C₁₋₇ alkoxy group, discussed below), aC₃₋₂₀ heterocyclyl group (also referred to as a C₃₋₂₀ heterocyclyloxygroup), or a C₅₋₂₀ aryl group (also referred to as a C₅₋₂₀ aryloxygroup), preferably a C₁₋₇ alkyl group.

Alkoxy: —OR, wherein R is an alkyl group, for example, a C₁₋₇ alkylgroup. Examples of C₁₋₇ alkoxy groups include, but are not limited to,—OMe (methoxy), —OEt (ethoxy), —O(nPr) (n-propoxy), —O(iPr)(isopropoxy), —O(nBu) (n-butoxy), —O(sBu) (sec-butoxy), —O(iBu)(isobutoxy), and —O(tBu) (tert-butoxy).

Carboxy (carboxylic acid): —C(═O)OH.

Ester (carboxylate, carboxylic acid ester, oxycarbonyl): —C(═O)OR,wherein R is an ester substituent, for example, a C1-7 alkyl group, aC₃₋₂₀ heterocyclyl group, or a C₅₋₂₀ aryl group, preferably a C₁₋₇ alkylgroup. Examples of ester groups include, but are not limited to,—C(═O)OCH₃, —C(═O)OCH₂CH₃, —C(═O)OC(CH₃)₃, and —C(═O)OPh.

Amino: —NR¹R², wherein R¹ and R² are independently amino substituents,for example, hydrogen, a C₁₋₇ alkyl group (also referred to as C₁₋₇alkylamino or di-C₁₋₇ alkylamino), a C₃₋₂₀ heterocyclyl group, or aC₅₋₂₀ aryl group, preferably H or a alkyl group, or, in the case of a“cyclic” amino group, R¹ and R², taken together with the nitrogen atomto which they are attached, form a heterocyclic ring having from 4 to 8ring atoms. Amino groups may be primary (—NH₂), secondary (—NHR¹), ortertiary (—NHR¹R²), and in cationic form, may be quaternary (—⁺NR¹R²R³).Examples of amino groups include, but are not limited to, —NH₂, —NHCH₃,—NHC(CH₃)₂, —N(CH₃)₂, —N(CH₂CH₃)₂, and —NHPh. Examples of cyclic aminogroups include, but are not limited to, aziridino, azetidino,pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.

Amido (carbamoyl, carbamyl, aminocarbonyl, carboxamide): —C(═O)NR¹R²,wherein R¹ and R² are independently amino substituents, as defined foramino groups. Examples of amido groups include, but are not limited to,—C(═O)NH₂, —C(═O)NHCH₃, —C(═O)N(CH₃)₂, —C(═O)NHCH₂CH₃, and—C(═O)N(CH₂CH₃)₂, as well as amido groups in which R¹ and R², togetherwith the nitrogen atom to which they are attached, form a heterocyclicstructure as in, for example, piperidinocarbonyl, morpholinocarbonyl,thiomorpholinocarbonyl, and piperazinocarbonyl.

Nitro: —NO₂.

Azido: —N₃.

Cyano (nitrile, carbonitrile): —CN.

Antibody

Anti-DLK1

In one aspect the antibody is an antibody that binds to DLK1.

Delta-like 1 homolog protein (DLK-1) is an EGF-like membrane boundprotein consisting of six tandem EGF-like repeats, a juxtamembraneregion with a TACE (ADAM17)-mediated cleavage site, a transmembranedomain, and a short intracellular tail. DLK-1 is strongly expressedduring fetal development, but its expression is turned down and highlyrestricted in adults. Conversely, DLK-1 gets re-expressed in severaltumors, such as neuroblastoma, hepatocellular carcinoma (HCC),rhabdomyosarcoma, small cell lung cancer, myelodysplastic syndrome andacute myeloid leukemia. Interestingly, in HCC DLK-1 has been shown to bea marker of cancer stem cells, a subpopulation of cells responsible fortumor initiation, growth, metastasis, and recurrence.

Altogether, DLK-1 represents an attractive target for an antibody-drugconjugate (ADC) approach based on its selective expression in a widerange of malignancies and restricted expression in healthy organs, aswell as its expression on HCC cancer stem cells.

HuBa-1-3d

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.7. In some embodiments the VHdomain further comprises a VH CDR2 with the amino acid sequence of SEQID NO.6, and/or a VH CDR1 with the amino acid sequence of SEQ ID NO.5.In some embodiments the antibody comprises a VH domain having a VH CDR1with the amino acid sequence of SEQ ID NO.5, a VH CDR2 with the aminoacid sequence of SEQ ID NO.6, and a VH CDR3 with the amino acid sequenceof SEQ ID NO.7. In preferred embodiments the antibody comprises a VHdomain having the sequence according to SEQ ID NO. 1.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.10. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.9, and/ora VL CDR1 with the amino acid sequence of SEQ ID NO.8. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.8, a VL CDR2 with the amino acidsequence of SEQ ID NO.9, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.10. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 2.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.1 and theVL domain comprises the sequence of SEQ ID NO.2.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds DLK1.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.1 paired with SEQ ID NO.2.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 3 paired with a light chain having the sequenceof SEQ ID NO.4. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.3, eachpaired with a light chain having the sequence of SEQ ID NO.4.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 11 paired with a light chain having the sequenceof SEQ ID NO.4. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.11, eachpaired with a light chain having the sequence of SEQ ID NO.4.

In one aspect the antibody is an antibody as described herein which hasbeen modified (or further modified) as described below. In someembodiments the antibody is a humanised, deimmunised or resurfacedversion of an antibody disclosed herein.

Anti-KAAG1

In one aspect the antibody is an antibody that binds to KAAG1.

Antibody 3A4

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.107. In some embodiments theVH domain further comprises a VH CDR2 with the amino acid sequence ofSEQ ID NO.106, and/or a VH CDR1 with the amino acid sequence of SEQ IDNO.105. In some embodiments the antibody comprises a VH domain having aVH CDR1 with the amino acid sequence of SEQ ID NO.105, a VH CDR2 withthe amino acid sequence of SEQ ID NO.106, and a VH CDR3 with the aminoacid sequence of SEQ ID NO.107. In preferred embodiments the antibodycomprises a VH domain having the sequence according to SEQ ID NO. 101.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.110. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.109,and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.108. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.108, a VL CDR2 with the amino acidsequence of SEQ ID NO.109, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.110. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 102, SEQ ID NO.113,or SEQ ID NO.115.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.101 andthe VL domain comprises the sequence of SEQ ID NO.102, SEQ ID NO.113, orSEQ ID NO.115.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds KAAG1.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.101 paired with SEQ ID NO.102, SEQ ID NO.113, or SEQ IDNO.115.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 103 paired with a light chain having the sequenceof SEQ ID NO.104, SEQ ID NO.114, or SEQ ID NO.116. In some embodimentsthe antibody is an intact antibody comprising two heavy chains havingthe sequence of SEQ ID NO.103, each paired with a light chain having thesequence of SEQ ID NO.104, SEQ ID NO.114, or SEQ ID NO.116.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 111 paired with a light chain having the sequenceof SEQ ID NO.104, SEQ ID NO.114, or SEQ ID NO.116. In some embodimentsthe antibody is an intact antibody comprising two heavy chains havingthe sequence of SEQ ID NO.111, each paired with a light chain having thesequence of SEQ ID NO.104, SEQ ID NO.114, or SEQ ID NO.116.

In one aspect the antibody is an antibody as described herein which hasbeen modified (or further modified) as described below. In someembodiments the antibody is a humanised, deimmunised or resurfacedversion of an antibody disclosed herein.

Anti-Mesothelin

In one aspect the antibody is an antibody that binds to Mesothelin.

ADCT-XA4

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.207. In some embodiments theVH domain further comprises a VH CDR2 with the amino acid sequence ofSEQ ID NO.206, and/or a VH CDR1 with the amino acid sequence of SEQ IDNO.205. In some embodiments the antibody comprises a VH domain having aVH CDR1 with the amino acid sequence of SEQ ID NO.205, a VH CDR2 withthe amino acid sequence of SEQ ID NO.206, and a VH CDR3 with the aminoacid sequence of SEQ ID NO.207. In preferred embodiments the antibodycomprises a VH domain having the sequence according to SEQ ID NO. 201.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.210. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.209,and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.208. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.208, a VL CDR2 with the amino acidsequence of SEQ ID NO.209, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.210. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 202.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.201 andthe VL domain comprises the sequence of SEQ ID NO.202.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds Mesothelin.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.201 paired with SEQ ID NO.202.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 203 paired with a light chain having the sequenceof SEQ ID NO.204. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.203, eachpaired with a light chain having the sequence of SEQ ID NO.204.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 211 paired with a light chain having the sequenceof SEQ ID NO.204. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.211, eachpaired with a light chain having the sequence of SEQ ID NO.204.

ADCT-XFT

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.218. In some embodiments theVH domain further comprises a VH CDR2 with the amino acid sequence ofSEQ ID NO.217, and/or a VH CDR1 with the amino acid sequence of SEQ IDNO.216. In some embodiments the antibody comprises a VH domain having aVH CDR1 with the amino acid sequence of SEQ ID NO.216, a VH CDR2 withthe amino acid sequence of SEQ ID NO.217, and a VH CDR3 with the aminoacid sequence of SEQ ID NO.218. In preferred embodiments the antibodycomprises a VH domain having the sequence according to SEQ ID NO. 212.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.221. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.220,and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.219. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.219, a VL CDR2 with the amino acidsequence of SEQ ID NO.220, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.221. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 213.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.212 andthe VL domain comprises the sequence of SEQ ID NO.213.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds Mesothelin.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.212 paired with SEQ ID NO.213.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 214 paired with a light chain having the sequenceof SEQ ID NO.215. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.214, eachpaired with a light chain having the sequence of SEQ ID NO.215.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 222 paired with a light chain having the sequenceof SEQ ID NO.215. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.222, eachpaired with a light chain having the sequence of SEQ ID NO.215.

ADCT-X09

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.229. In some embodiments theVH domain further comprises a VH CDR2 with the amino acid sequence ofSEQ ID NO.228, and/or a VH CDR1 with the amino acid sequence of SEQ IDNO.227. In some embodiments the antibody comprises a VH domain having aVH CDR1 with the amino acid sequence of SEQ ID NO.227, a VH CDR2 withthe amino acid sequence of SEQ ID NO.228, and a VH CDR3 with the aminoacid sequence of SEQ ID NO.229. In preferred embodiments the antibodycomprises a VH domain having the sequence according to SEQ ID NO. 223.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.232. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.231,and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.230. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.230, a VL CDR2 with the amino acidsequence of SEQ ID NO.231, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.232. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 224.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.223 andthe VL domain comprises the sequence of SEQ ID NO.224.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds Mesithelin.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.223 paired with SEQ ID NO.224.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 225 paired with a light chain having the sequenceof SEQ ID NO.226. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.225, eachpaired with a light chain having the sequence of SEQ ID NO.226.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 233 paired with a light chain having the sequenceof SEQ ID NO.226. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.233, eachpaired with a light chain having the sequence of SEQ ID NO.226.

ADCT-X09.2

In some embodiments the antibody comprises a VH domain having a VH CDR3with the amino acid sequence of SEQ ID NO.240. In some embodiments theVH domain further comprises a VH CDR2 with the amino acid sequence ofSEQ ID NO.239, and/or a VH CDR1 with the amino acid sequence of SEQ IDNO.238. In some embodiments the antibody comprises a VH domain having aVH CDR1 with the amino acid sequence of SEQ ID NO.238, a VH CDR2 withthe amino acid sequence of SEQ ID NO.239, and a VH CDR3 with the aminoacid sequence of SEQ ID NO.240. In preferred embodiments the antibodycomprises a VH domain having the sequence according to SEQ ID NO.234.

The antibody may further comprise a VL domain. In some embodiments theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.243. In some embodiments the VL domain furthercomprises a VL CDR2 with the amino acid sequence of SEQ ID NO.242,and/or a VL CDR1 with the amino acid sequence of SEQ ID NO.241. In someembodiments the antibody comprises a VL domain having a VL CDR1 with theamino acid sequence of SEQ ID NO.241, a VL CDR2 with the amino acidsequence of SEQ ID NO.242, and a VL CDR3 with the amino acid sequence ofSEQ ID NO.243. In preferred embodiments the antibody comprises a VLdomain having the sequence according to SEQ ID NO. 235.

In preferred embodiments the antibody comprises a VH domain and a VLdomain. Preferably the VH comprises the sequence of SEQ ID NO.234 andthe VL domain comprises the sequence of SEQ ID NO.235.

The VH and VL domain(s) may pair so as to form an antibody antigenbinding site that binds Mesothelin.

In some embodiments the antibody is an intact antibody comprising a VHdomain paired with a VL domain, the VH and VL domains having sequencesof SEQ ID NO.234 paired with SEQ ID NO.235.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 236 paired with a light chain having the sequenceof SEQ ID NO.237. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.236, eachpaired with a light chain having the sequence of SEQ ID NO.237.

In some embodiments the antibody comprises a heavy chain having thesequence of SEQ ID NO. 244 paired with a light chain having the sequenceof SEQ ID NO.237. In some embodiments the antibody is an intact antibodycomprising two heavy chains having the sequence of SEQ ID NO.244, eachpaired with a light chain having the sequence of SEQ ID NO.237.

Terminology

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), intact antibodies and antibody fragments, so long as theyexhibit the desired biological activity, for example, the ability tobind DLK1, KAAG1, or Mesothelin. Antibodies may be murine, human,humanized, chimeric, or derived from other species. An antibody is aprotein generated by the immune system that is capable of recognizingand binding to a specific antigen. (Janeway, C., Travers, P., Walport,M., Shlomchik (2001) Immuno Biology, 5th Ed., Garland Publishing, NewYork). A target antigen generally has numerous binding sites, alsocalled epitopes, recognized by CDRs on multiple antibodies. Eachantibody that specifically binds to a different epitope has a differentstructure. Thus, one antigen may have more than one correspondingantibody. An antibody includes a full-length immunoglobulin molecule oran immunologically active portion of a full-length immunoglobulinmolecule, i.e., a molecule that contains an antigen binding site thatimmunospecifically binds an antigen of a target of interest or partthereof, such targets including but not limited to, cancer cell or cellsthat produce autoimmune antibodies associated with an autoimmunedisease. The immunoglobulin can be of any type (e.g. IgG, IgE, IgM, IgD,and IgA), class (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) orsubclass, or allotype (e.g. human G1m1, G1m2, G1m3, non-G1m1 [that, isany allotype other than G1m1], G1m17, G2m23, G3m21, G3m28, G3m11, G3m5,G3m13, G3m14, G3m10, G3m15, G3m16, G3m6, G3m24, G3m26, G3m27, A2m1,A2m2, Km1, Km2 and Km3) of immunoglobulin molecule. The immunoglobulinscan be derived from any species, including human, murine, or rabbitorigin.

As used herein, “binds DLK1” is used to mean the antibody binds DLK1with a higher affinity than a non-specific partner such as Bovine SerumAlbumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1GI:3336842, record update date: Jan. 7, 2011 02:30 PM). In someembodiments the antibody binds DLK1 with an association constant (K_(a))at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000, 5000, 10⁴,10⁵ or 10⁶-fold higher than the antibody's association constant for BSA,when measured at physiological conditions. The antibodies of theinvention can bind DLK1 with a high affinity. For example, in someembodiments the antibody can bind DLK1 with a K_(D) equal to or lessthan about 10⁻⁶ M, such as 1×10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹,10⁻¹², 10⁻¹³ or 10⁻¹⁴.

DLK1 is member of the EGF-like family of homeotic proteins. In someembodiments, the DLK1 polypeptide corresponds to Genbank accession no.CAA78163, version no. CAA78163.1, record update date: Feb. 2, 2011 10:34AM (SEQ ID NO.12). In one embodiment, the nucleic acid encoding DLK1polypeptide corresponds to Genbank accession no. Z12172, version noZ12172.1, record update date: Feb. 2, 2011 10:34 AM. In someembodiments, the DLK1 polypeptide has the sequence of SEQ ID NO.13.

As used herein, “binds KAAG1” is used to mean the antibody binds KAAG1with a higher affinity than a non-specific partner such as Bovine SerumAlbumin (BSA, Genbank accession no. CAA76847, version no. CAA76847.1GI:3336842, record update date: Jan. 7, 2011 02:30 PM). In someembodiments the antibody binds KAAG1 with an association constant(K_(a)) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000, 2000,5000, 10⁴, 10⁵ or 10⁸-fold higher than the antibody's associationconstant for BSA, when measured at physiological conditions. Theantibodies of the invention can bind KAAG1 with a high affinity. Forexample, in some embodiments the antibody can bind KAAG1 with a K_(D)equal to or less than about 10⁻⁶ M, such as 1×10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹,10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹³ or 10⁻¹⁴.

KAAG1 (Kidney associated antigen 1) is expressed in testis and kidney,and, at lower levels, in urinary bladder and liver. It is expressed by ahigh proportion of tumors of various histologic origin, includingmelanomas, sarcomas and colorectal carcinomas. In some embodiments, theKAAG1 polypeptide corresponds to Genbank accession no. AAF23613, versionno. AAF23613.1. In one embodiment, the nucleic acid encoding KAAG1polypeptide corresponds to Genbank accession no. AF181722, version noAF181722.1. In some embodiments, the KAAG1 polypeptide has the sequenceof SEQ ID NO.112.

As used herein, “binds Mesothelin” is used to mean the antibody bindsMesothelin with a higher affinity than a non-specific partner such asBovine Serum Albumin (BSA, Genbank accession no. CAA76847, version no.CAA76847.1 GI:3336842, record update date: Jan. 7, 2011 02:30 PM). Insome embodiments the antibody binds Mesothelin with an associationconstant (K_(a)) at least 2, 3, 4, 5, 10, 20, 50, 100, 200, 500, 1000,2000, 5000, 10⁴, 10⁵ or 10⁶-fold higher than the antibody's associationconstant for BSA, when measured at physiological conditions. Theantibodies of the invention can bind Mesothelin with a high affinity.For example, in some embodiments the antibody can bind Mesothelin with aK_(D) equal to or less than about 10⁻⁶ M, such as 1×10⁻⁶, 10⁻⁷, 10⁻⁸,10³¹ ⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10³¹ ¹³ or 10⁻¹⁴.

Mesothelin is a 40 kDa protein present on normal mesothelial cells. Themesothelin gene encodes a precursor protein that is processed to yieldmesothelin, which is attached to the cell membrane by aglycophosphatidylinositol linkage, and a 31-kDa shed fragment namedmegakaryocyte-potentiating factor (MPF). It has been proposed thatmesothelin may be involved in cell adhesion. In some embodiments, theMesothelin polypeptide corresponds to Genbank accession no. AAC50348,version no. AAC50348.1, record update date: Jun. 23, 2010 09:12 AM. Inone embodiment, the nucleic acid encoding Mesothelin polypeptidecorresponds to Genbank accession no. U40434, version no U40434.1, recordupdate date: Jun. 23, 2010 09:12 AM. In some embodiments, the Mesothelinpolypeptide has the sequence of SEQ ID NO.245. In some embodiments, theMesothelin polypeptide has the sequence of SEQ ID NO.246.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and scFv fragments;diabodies; linear antibodies; fragments produced by a Fab expressionlibrary, anti-idiotypic (anti-Id) antibodies, CDR (complementarydetermining region), and epitope-binding fragments of any of the abovewhich immunospecifically bind to cancer cell antigens, viral antigens ormicrobial antigens, single-chain antibody molecules; and multispecificantibodies formed from antibody fragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies, i.e.the individual antibodies comprising the population are identical exceptfor possible naturally occurring mutations that may be present in minoramounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see, U.S. Pat. No. 4,816,567). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al. (1991) Nature, 352:624-628;Marks et al. (1991) J. Mol. Biol., 222:581-597 or from transgenic micecarrying a fully human immunoglobulin system (Lonberg (2008) Curr.Opinion 20(4):450-459).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodiesinclude “primatized” antibodies comprising variable domainantigen-binding sequences derived from a non-human primate (e.g. OldWorld Monkey or Ape) and human constant region sequences.

An “intact antibody” herein is one comprising VL and VH domains, as wellas a light chain constant domain (CL) and heavy chain constant domains,CH1, CH2 and CH3. The constant domains may be native sequence constantdomains (e.g. human native sequence constant domains) or amino acidsequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc region (a native sequence Fc region or amino acidsequence variant Fc region) of an antibody. Examples of antibodyeffector functions include C1q binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

Depending on the amino acid sequence of the constant domain of theirheavy chains, intact antibodies can be assigned to different “classes.”There are five major classes of intact antibodies: IgA, IgD, IgE, IgG,and IgM, and several of these may be further divided into “subclasses”(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains that correspond to the different classes of antibodiesare called α, δ, ε, γ, and μ, respectively. The subunit structures andthree-dimensional configurations of different classes of immunoglobulinsare well known.

Modification of Antibodies

The antibodies disclosed herein may be modified. For example, to makethem less immunogenic to a human subject. This may be achieved using anyof a number of techniques familiar to the person skilled in the art.Some of these techniques are described in more detail below.

Humanisation

Techniques to reduce the in vivo immunogenicity of a non-human antibodyor antibody fragment include those termed “humanisation”.

A “humanized antibody” refers to a polypeptide comprising at least aportion of a modified variable region of a human antibody wherein aportion of the variable region, preferably a portion substantially lessthan the intact human variable domain, has been substituted by thecorresponding sequence from a non-human species and wherein the modifiedvariable region is linked to at least another part of another protein,preferably the constant region of a human antibody. The expression“humanized antibodies” includes human antibodies in which one or morecomplementarity determining region (“CDR”) amino acid residues and/orone or more framework region (“FW” or “FR”) amino acid residues aresubstituted by amino acid residues from analogous sites in rodent orother non-human antibodies. The expression “humanized antibody” alsoincludes an immunoglobulin amino acid sequence variant or fragmentthereof that comprises an FR having substantially the amino acidsequence of a human immunoglobulin and a CDR having substantially theamino acid sequence of a non-human immunoglobulin.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. Or, looked at another way, a humanized antibody is ahuman antibody that also contains selected sequences from non-human(e.g. murine) antibodies in place of the human sequences. A humanizedantibody can include conservative amino acid substitutions ornon-natural residues from the same or different species that do notsignificantly alter its binding and/or biologic activity. Suchantibodies are chimeric antibodies that contain minimal sequence derivedfrom non-human immunoglobulins.

There are a range of humanisation techniques, including ‘CDR grafting’,‘guided selection’, ‘deimmunization’, ‘resurfacing’ (also known asVeneering′), ‘composite antibodies’, ‘Human String Content Optimisation’and framework shuffling.

CDR Grafting

In this technique, the humanized antibodies are human immunoglobulins(recipient antibody) in which residues from a complementary-determiningregion (CDR) of the recipient antibody are replaced by residues from aCDR of a non-human species (donor antibody) such as mouse, rat, camel,bovine, goat, or rabbit having the desired properties (in effect, thenon-human CDRs are ‘grafted’ onto the human framework). In someinstances, framework region (FR) residues of the human immunoglobulinare replaced by corresponding non-human residues (this may happen when,for example, a particular FR residue has significant effect on antigenbinding).

Furthermore, humanized antibodies can comprise residues that are foundneither in the recipient antibody nor in the imported CDR or frameworksequences. These modifications are made to further refine and maximizeantibody performance. Thus, in general, a humanized antibody willcomprise all of at least one, and in one aspect two, variable domains,in which all or all of the hypervariable loops correspond to those of anon-human immunoglobulin and all or substantially all of the FR regionsare those of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), or that of a human immunoglobulin.

Guided Selection

The method consists of combining the V_(H) or V_(L) domain of a givennon-human antibody specific for a particular epitope with a human V_(H)or V_(L) library and specific human V domains are selected against theantigen of interest. This selected human VH is then combined with a VLlibrary to generate a completely human VH×VL combination. The method isdescribed in Nature Biotechnology (N.Y.) 12, (1994) 899-903.

Composite Antibodies

In this method, two or more segments of amino acid sequence from a humanantibody are combined within the final antibody molecule. They areconstructed by combining multiple human VH and VL sequence segments incombinations which limit or avoid human T cell epitopes in the finalcomposite antibody V regions. Where required, T cell epitopes arelimited or avoided by, exchanging V region segments contributing to orencoding a T cell epitope with alternative segments which avoid T cellepitopes. This method is described in US 2008/0206239 A1.

Deimmunization

This method involves the removal of human (or other second species)T-cell epitopes from the V regions of the therapeutic antibody (or othermolecule). The therapeutic antibodies V-region sequence is analysed forthe presence of MHC class II-binding motifs by, for example, comparisonwith databases of MHC-binding motifs (such as the “motifs” databasehosted at www.wehi.edu.au). Alternatively, MHC class II-binding motifsmay be identified using computational threading methods such as thosedevised by Altuvia et al. (J. Mol. Biol. 249 244-250 (1995)); in thesemethods, consecutive overlapping peptides from the V-region sequencesare testing for their binding energies to MHC class II proteins. Thisdata can then be combined with information on other sequence featureswhich relate to successfully presented peptides, such as amphipathicity,Rothbard motifs, and cleavage sites for cathepsin B and other processingenzymes.

Once potential second species (e.g. human) T-cell epitopes have beenidentified, they are eliminated by the alteration of one or more aminoacids. The modified amino acids are usually within the T-cell epitopeitself, but may also be adjacent to the epitope in terms of the primaryor secondary structure of the protein (and therefore, may not beadjacent in the primary structure). Most typically, the alteration is byway of substitution but, in some circumstances amino acid addition ordeletion will be more appropriate.

All alterations can be accomplished by recombinant DNA technology, sothat the final molecule may be prepared by expression from a recombinanthost using well established methods such as Site Directed Mutagenesis.However, the use of protein chemistry or any other means of molecularalteration is also possible.

Resurfacing

This method involves:

-   -   (a) determining the conformational structure of the variable        region of the non-human (e.g. rodent) antibody (or fragment        thereof) by constructing a three-dimensional model of the        non-human antibody variable region;    -   (b) generating sequence alignments using relative accessibility        distributions from x-ray crystallographic structures of a        sufficient number of non-human and human antibody variable        region heavy and light chains to give a set of heavy and light        chain framework positions wherein the alignment positions are        identical in 98% of the sufficient number of non-human antibody        heavy and light chains;    -   (c) defining for the non-human antibody to be humanized, a set        of heavy and light chain surface exposed amino acid residues        using the set of framework positions generated in step (b);    -   (d) identifying from human antibody amino acid sequences a set        of heavy and light chain surface exposed amino acid residues        that is most closely identical to the set of surface exposed        amino acid residues defined in step (c), wherein the heavy and        light chain from the human antibody are or are not naturally        paired;    -   (e) substituting, in the amino acid sequence of the non-human        antibody to be humanized, the set of heavy and light chain        surface exposed amino acid residues defined in step (c) with the        set of heavy and light chain surface exposed amino acid residues        identified in step (d);    -   (f) constructing a three-dimensional model of the variable        region of the non-human antibody resulting from the substituting        specified in step (e);    -   (g) identifying, by comparing the three-dimensional models        constructed in steps (a) and (f), any amino acid residues from        the sets identified in steps (c) or (d), that are within 5        Angstroms of any atom of any residue of the complementarity        determining regions of the non-human antibody to be humanized;        and    -   (h) changing any residues identified in step (g) from the human        to the original non-human amino acid residue to thereby define a        non-human antibody humanizing set of surface exposed amino acid        residues; with the proviso that step (a) need not be conducted        first, but must be conducted prior to step (g).

Superhumanization

The method compares the non-human sequence with the functional humangermline gene repertoire. Those human genes encoding canonicalstructures identical or closely related to the non-human sequences areselected. Those selected human genes with highest homology within theCDRs are chosen as FR donors. Finally, the non-human CDRs are graftedonto these human FRs. This method is described in patent WO 2005/079479A2.

Human String Content Optimization

This method compares the non-human (e.g. mouse) sequence with therepertoire of human germline genes and the differences are scored asHuman String Content (HSC) that quantifies a sequence at the level ofpotential MHC/T-cell epitopes. The target sequence is then humanized bymaximizing its HSC rather than using a global identity measure togenerate multiple diverse humanized variants (described in MolecularImmunology, 44, (2007) 1986-1998).

Framework Shuffling

The CDRs of the non-human antibody are fused in-frame to cDNA poolsencompassing all known heavy and light chain human germline geneframeworks. Humanised antibodies are then selected by e.g. panning ofthe phage displayed antibody library. This is described in Methods 36,43-60 (2005).

Modification of Antibody with Azide

The antibody may prepared for conjugation with the drug linker through athree step process:

-   -   (1) Expression of antibody (Ab) bearing the core N-glycan in a        suitable expression system (e.g. a CHO cell line). The core        N-glycan is typically conjugated to Asn-297 of the heavy chain        according to the numbering system of Kabat;    -   (2) trimming of all glycan isoforms (complex, hybrid,        high-mannose) with an endoglycosidase to leave the core GlcNAc;        and    -   (3) enzymatic transfer to the core GlcNAc of a N-acetylgalactose        residue harboring an azide group for conjugation to the drug        linker.

An overview of the above process is set out in van Geel, R., et al.,Bioconjugate Chemistry, 2015, 26, 2233-2242; DOI:10.1021/acs.bioconjchem.5b00224. Alternatively, a one-pot process may beused—see the examples.

Embodiments

X

In some embodiments, X is a single bond.

In other embodiments, X is —CH₂—.

In further embodiments, X is —C₂H₄—.

In some embodiments, n is 1 to 4.

In some of these embodiments, n is 1.

In other of these embodiments, n is 2.

In further of these embodiments, n is 4.

R⁷

In one embodiment, R⁷ is methyl.

In another embodiment, R⁷ is phenyl.

R²

When there is a double bond present between C2 and C3, R² is selectedfrom: (a) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene;

(b) C₁₋₅ saturated aliphatic alkyl;

(c) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl.

When R² is a C₅₋₁₀ aryl group, it may be a C₅₋₇ aryl group. A C₅₋₇ arylgroup may be a phenyl group or a C₅₋₇ heteroaryl group, for examplefuranyl, thiophenyl and pyridyl. In some embodiments, R² is preferablyphenyl. In other embodiments, R¹² is preferably thiophenyl, for example,thiophen-2-yl and thiophen-3-yl.

When R² is a C₅₋₁₀ aryl group, it may be a C₈₋₁₀ aryl, for example aquinolinyl or isoquinolinyl group. The quinolinyl or isoquinolinyl groupmay be bound to the PBD core through any available ring position. Forexample, the quinolinyl may be quinolin-2-yl, quinolin-3-yl,quinolin-4yl, quinolin-5-yl, quinolin-6-yl, quinolin-7-yl andquinolin-8-yl. Of these quinolin-3-yl and quinolin-6-yl may bepreferred. The isoquinolinyl may be isoquinolin-1-yl, isoquinolin-3-yl,isoquinolin-4yl, isoquinolin-5-yl, isoquinolin-6-yl, isoquinolin-7-yland isoquinolin-8-yl. Of these isoquinolin-3-yl and isoquinolin-6-yl maybe preferred.

When R² is a C₅₋₁₀ aryl group, it may bear any number of substituentgroups. It preferably bears from 1 to 3 substituent groups, with 1 and 2being more preferred, and singly substituted groups being mostpreferred. The substituents may be any position.

Where R² is C₅₋₇ aryl group, a single substituent is preferably on aring atom that is not adjacent the bond to the remainder of thecompound, i.e. it is preferably β or γ to the bond to the remainder ofthe compound. Therefore, where the C₅₋₇ aryl group is phenyl, thesubstituent is preferably in the meta- or para-positions, and morepreferably is in the para-position.

Where R² is a C₈₋₁₀ aryl group, for example quinolinyl or isoquinolinyl,it may bear any number of substituents at any position of the quinolineor isoquinoline rings. In some embodiments, it bears one, two or threesubstituents, and these may be on either the proximal and distal ringsor both (if more than one substituent).

R² substituents, when R² is a C₅₋₁₀ aryl group

If a substituent on R² when R² is a C₅₋₁₀ aryl group is halo, it ispreferably F or Cl, more preferably Cl.

If a substituent on R² when R² is a C₅₋₁₀ aryl group is ether, it may insome embodiments be an alkoxy group, for example, a C₁₋₇ alkoxy group(e.g. methoxy, ethoxy) or it may in some embodiments be a C₅₋₇ aryloxygroup (e.g. phenoxy, pyridyloxy, furanyloxy). The alkoxy group mayitself be further substituted, for example by an amino group (e.g.dimethylamino).

If a substituent on R² when R² is a C₅₋₁₀ aryl group is C₁₋₇ alkyl, itmay preferably be a C₁₋₄ alkyl group (e.g. methyl, ethyl, propryl,butyl).

If a substituent on R² when R² is a C₅₋₁₀ aryl group is C₃₋₇heterocyclyl, it may in some embodiments be C₆ nitrogen containingheterocyclyl group, e.g. morpholino, thiomorpholino, piperidinyl,piperazinyl. These groups may be bound to the rest of the PBD moiety viathe nitrogen atom. These groups may be further substituted, for example,by C₁₋₄ alkyl groups. If the C₆ nitrogen containing heterocyclyl groupis piperazinyl, the said further substituent may be on the secondnitrogen ring atom.

If a substituent on R² when R² is a C5-10 aryl group is bis-oxy-C₁₋₃alkylene, this is preferably bis-oxy-methylene or bis-oxy-ethylene.

If a substituent on R² when R² is a C₅₋₁₀ aryl group is ester, this ispreferably methyl ester or ethyl ester.

Particularly preferred substituents when R² is a C5-10 aryl groupinclude methoxy, ethoxy, fluoro, chloro, cyano, bis-oxy-methylene,methyl-piperazinyl, morpholino and methyl-thiophenyl. Other particularlypreferred substituent for R² are dimethylaminopropyloxy and carboxy.

Particularly preferred substituted R² groups when R² is a C₅₋₁₀ arylgroup include, but are not limited to, 4-methoxy-phenyl,3-methoxyphenyl, 4-ethoxy-phenyl, 3-ethoxy-phenyl, 4-fluoro-phenyl,4-chloro-phenyl, 3,4-bisoxymethylene-phenyl, 4-methylthiophenyl,4-cyanophenyl, 4-phenoxyphenyl, quinolin-3-yl and quinolin-6-yl,isoquinolin-3-yl and isoquinolin-6-yl, 2-thienyl, 2-furanyl,methoxynaphthyl, and naphthyl. Another possible substituted R² group is4-nitrophenyl. R² groups of particular interest include4-(4-methylpiperazin-1-yl)phenyl and 3,4-bisoxymethylene-phenyl.

When R² is C₁₋₅ saturated aliphatic alkyl, it may be methyl, ethyl,propyl, butyl or pentyl. In some embodiments, it may be methyl, ethyl orpropyl (n-pentyl or isopropyl). In some of these embodiments, it may bemethyl. In other embodiments, it may be butyl or pentyl, which may belinear or branched.

When R² is C₃₋₆ saturated cycloalkyl, it may be cyclopropyl, cyclobutyl,cyclopentyl or cyclohexyl. In some embodiments, it may be cyclopropyl.

When R² is

each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5. In someembodiments, the total number of carbon atoms in the R² group is no morethan 4 or no more than 3.

In some embodiments, one of R²¹, R²² and R²³ is H, with the other twogroups being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In other embodiments, two of R²¹, R²² and R²³ are H, with the othergroup being selected from H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃alkynyl and cyclopropyl.

In some embodiments, the groups that are not H are selected from methyland ethyl. In some of these embodiments, the groups that are not H aremethyl.

In some embodiments, R²¹ is H.

In some embodiments, R²² is H.

In some embodiments, R²³ is H.

In some embodiments, R²¹ and R²² are H.

In some embodiments, R²¹ and R²³ are H.

In some embodiments, R²² and R²³ are H.

A R² group of particular interest is:

When R² is

one of R^(25a) and R^(25b) is H and the other is selected from: phenyl,which phenyl is optionally substituted by a group selected from halo,methyl, methoxy;

pyridyl; and thiophenyl. In some embodiments, the group which is not His optionally substituted phenyl. If the phenyl optional substituent ishalo, it is preferably fluoro. In some embodiment, the phenyl group isunsubstituted.

When R² is

R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo methyl, methoxy; pyridyl; and thiophenyl. Ifthe phenyl optional substituent is halo, it is preferably fluoro. Insome embodiment, the phenyl group is unsubstituted.

In some embodiments, R²⁴ is selected from H, methyl, ethyl, ethenyl andethynyl. In some of these embodiments, R²⁴ is selected from H andmethyl.

When there is a single bond present between C2 and C3,

R² is

where R^(26a) and R^(26b) are independently selected from H, F, C1-4saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C1-4 alkyl ester.

In some embodiments, it is preferred that R^(26a) and R^(26b) are bothH.

In other embodiments, it is preferred that R^(26a) and R^(26b) are bothmethyl.

In further embodiments, it is preferred that one of R^(26a) and R^(26b)is H, and the other is selected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl,which alkyl and alkenyl groups are optionally substituted. In thesefurther embodiment, it may be further preferred that the group which isnot H is selected from methyl and ethyl.

R¹²

The above preferences for R² apply equally to R¹².

In one embodiment of the invention, DL is

Drug Loading

The drug loading is the average number of PBD drugs per antibody, e.g.antibody.

The average number of drugs per antibody in preparations of ADC fromconjugation reactions may be characterized by conventional means such asUV, reverse phase HPLC, HIC, mass spectroscopy, ELISA assay, andelectrophoresis. The quantitative distribution of ADC in terms of p mayalso be determined. By ELISA, the averaged value of p in a particularpreparation of ADC may be determined (Hamblen et al (2004) Clin. CancerRes. 10:7063-7070; Sanderson et al (2005) Clin. Cancer Res. 11:843-852).However, the distribution of p (drug) values is not discernible by theantibody-antigen binding and detection limitation of ELISA. Also, ELISAassay for detection of antibody-drug conjugates does not determine wherethe drug moieties are attached to the antibody, such as the heavy chainor light chain fragments, or the particular amino acid residues. In someinstances, separation, purification, and characterization of homogeneousADC where p is a certain value from ADC with other drug loadings may beachieved by means such as reverse phase HPLC or electrophoresis. Suchtechniques are also applicable to other types of conjugates.

For the present antibody-drug conjugates, p is limited by the number ofattachment sites on the antibody, i.e. the number of azide groups. Forexample, the antibody may have only one or two azide groups to which thedrug linker may be attached.

Typically, fewer than the theoretical maximum of drug moieties areconjugated to an antibody during a conjugation reaction. The loading(drug/antibody ratio) of an ADC may be controlled in several differentmanners, including: (i) limiting the molar excess of drug-linkerintermediate (D-L) or linker reagent relative to antibody, and (ii)limiting the conjugation reaction time or temperature.

Where more than one nucleophilic or electrophilic group of the antibodyreacts with a drug-linker intermediate, or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of drug moieties attached to an antibody,e.g. 1, 2, 3, etc. Liquid chromatography methods such as polymericreverse phase (PLRP) and hydrophobic interaction (HIC) may separatecompounds in the mixture by drug loading value. Preparations of ADC witha single drug loading value (p) may be isolated, however, these singleloading value ADCs may still be heterogeneous mixtures because the drugmoieties may be attached, via the linker, at different sites on theantibody.

Thus the antibody-drug conjugate compositions of the invention includemixtures of antibody-drug conjugate compounds where the antibody has oneor more PBD drug moieties and where the drug moieties may be attached tothe antibody at various amino acid residues.

In one embodiment, the average number of dimer pyrrolobenzodiazepinegroups per antibody is in the range 1 to 8. In some embodiments therange is selected from 1 to 4, 1 to 4, 2 to 4, and 1 to 3.

In some embodiments, there are one or two dimer pyrrolobenzodiazepinegroups per antibody.

Includes Other Forms

Unless otherwise specified, included in the above are the well knownionic, salt, solvate, and protected forms of these substituents. Forexample, a reference to carboxylic acid (—COOH) also includes theanionic (carboxylate) form (—COO⁻), a salt or solvate thereof, as wellas conventional protected forms. Similarly, a reference to an aminogroup includes the protonated form (—H⁺HR¹R²), a salt or solvate of theamino group, for example, a hydrochloride salt, as well as conventionalprotected forms of an amino group. Similarly, a reference to a hydroxylgroup also includes the anionic form (—O⁻), a salt or solvate thereof,as well as conventional protected forms.

Salts

It may be convenient or desirable to prepare, purify, and/or handle acorresponding salt of the active compound, for example, apharmaceutically-acceptable salt. Examples of pharmaceuticallyacceptable salts are discussed in Berge, et al., J. Pharm. Sci., 66,1-19 (1977).

For example, if the compound is anionic, or has a functional group whichmay be anionic (e.g. —COOH may be —COO⁻), then a salt may be formed witha suitable cation. Examples of suitable inorganic cations include, butare not limited to, alkali metal ions such as Na⁺ and K⁺, alkaline earthcations such as Ca²⁺ and Mg²⁺, and other cations such as Al⁺³. Examplesof suitable organic cations include, but are not limited to, ammoniumion (i.e. NH₄ ⁺) and substituted ammonium ions (e.g. NH₃R⁺, NH₂R₂ ⁺,NHR₃ ⁺, NR₄ ⁺). Examples of some suitable substituted ammonium ions arethose derived from: ethylamine, diethylamine, dicyclohexylamine,triethylamine, butylamine, ethylenediamine, ethanolamine,diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline,meglumine, and tromethamine, as well as amino acids, such as lysine andarginine. An example of a common quaternary ammonium ion is N(CH₃)₄ ⁺.

If the compound is cationic, or has a functional group which may becationic (e.g. —NH₂ may be —NH₃ ⁺), then a salt may be formed with asuitable anion. Examples of suitable inorganic anions include, but arenot limited to, those derived from the following inorganic acids:hydrochloric, hydrobromic, hydroiodic, sulfuric, sulfurous, nitric,nitrous, phosphoric, and phosphorous.

Examples of suitable organic anions include, but are not limited to,those derived from the following organic acids: 2-acetyoxybenzoic,acetic, ascorbic, aspartic, benzoic, camphorsulfonic, cinnamic, citric,edetic, ethanedisulfonic, ethanesulfonic, fumaric, glucheptonic,gluconic, glutamic, glycolic, hydroxymaleic, hydroxynaphthalenecarboxylic, isethionic, lactic, lactobionic, lauric, maleic, malic,methanesulfonic, mucic, oleic, oxalic, palmitic, pamoic, pantothenic,phenylacetic, phenylsulfonic, propionic, pyruvic, salicylic, stearic,succinic, sulfanilic, tartaric, toluenesulfonic, trifluoroacetic acidand valeric. Examples of suitable polymeric organic anions include, butare not limited to, those derived from the following polymeric acids:tannic acid, carboxymethyl cellulose.

Solvates

It may be convenient or desirable to prepare, purify, and/or handle acorresponding solvate of the active compound. The term “solvate” is usedherein in the conventional sense to refer to a complex of solute (e.g.active compound, salt of active compound) and solvent. If the solvent iswater, the solvate may be conveniently referred to as a hydrate, forexample, a mono-hydrate, a di-hydrate, a tri-hydrate, etc.

The invention includes compounds where a solvent adds across the iminebond of the PBD moiety, which is illustrated below where the solvent iswater or an alcohol (R^(A)OH, where R^(A) is C₁₋₄ alkyl):

These forms can be called the carbinolamine and carbinolamine etherforms of the PBD (as described in the section relating to R¹⁰ above).The balance of these equilibria depend on the conditions in which thecompounds are found, as well as the nature of the moiety itself.

These particular compounds may be isolated in solid form, for example,by lyophilisation.

Isomers

Certain compounds of the invention may exist in one or more particulargeometric, optical, enantiomeric, diasteriomeric, epimeric, atropic,stereoisomeric, tautomeric, conformational, or anomeric forms, includingbut not limited to, cis- and trans-forms; E- and Z-forms; c-, t-, andr-forms; endo- and exo-forms; R-, S-, and meso-forms; D- and L-forms; d-and I-forms; (+) and (−) forms; keto-, enol-, and enolate-forms; syn-and anti-forms; synclinal- and anticlinal-forms; α- and β-forms; axialand equatorial forms; boat-, chair-, twist-, envelope-, andhalfchair-forms; and combinations thereof, hereinafter collectivelyreferred to as “isomers” (or “isomeric forms”).

The term “chiral” refers to molecules which have the property ofnon-superimposability of the mirror image partner, while the term“achiral” refers to molecules which are superimposable on their mirrorimage partner.

The term “stereoisomers” refers to compounds which have identicalchemical constitution, but differ with regard to the arrangement of theatoms or groups in space.

“Diastereomer” refers to a stereoisomer with two or more centers ofchirality and whose molecules are not mirror images of one another.Diastereomers have different physical properties, e.g. melting points,boiling points, spectral properties, and reactivities. Mixtures ofdiastereomers may separate under high resolution analytical proceduressuch as electrophoresis and chromatography.

“Enantiomers” refer to two stereoisomers of a compound which arenon-superimposable mirror images of one another.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,“Stereochemistry of Organic Compounds”, John Wiley & Sons, Inc., NewYork, 1994. The compounds of the invention may contain asymmetric orchiral centers, and therefore exist in different stereoisomeric forms.It is intended that all stereoisomeric forms of the compounds of theinvention, including but not limited to, diastereomers, enantiomers andatropisomers, as well as mixtures thereof such as racemic mixtures, formpart of the present invention. Many organic compounds exist in opticallyactive forms, i.e., they have the ability to rotate the plane ofplane-polarized light. In describing an optically active compound, theprefixes D and L, or R and S, are used to denote the absoluteconfiguration of the molecule about its chiral center(s). The prefixes dand I or (+) and (−) are employed to designate the sign of rotation ofplane-polarized light by the compound, with (−) or I meaning that thecompound is levorotatory. A compound prefixed with (+) or d isdextrorotatory. For a given chemical structure, these stereoisomers areidentical except that they are mirror images of one another. A specificstereoisomer may also be referred to as an enantiomer, and a mixture ofsuch isomers is often called an enantiomeric mixture. A 50:50 mixture ofenantiomers is referred to as a racemic mixture or a racemate, which mayoccur where there has been no stereoselection or stereospecificity in achemical reaction or process. The terms “racemic mixture” and “racemate”refer to an equimolar mixture of two enantiomeric species, devoid ofoptical activity.

Note that, except as discussed below for tautomeric forms, specificallyexcluded from the term “isomers”, as used herein, are structural (orconstitutional) isomers (i.e. isomers which differ in the connectionsbetween atoms rather than merely by the position of atoms in space). Forexample, a reference to a methoxy group, —OCH₃, is not to be construedas a reference to its structural isomer, a hydroxymethyl group, —CH₂OH.Similarly, a reference to ortho-chlorophenyl is not to be construed as areference to its structural isomer, meta-chlorophenyl. However, areference to a class of structures may well include structurallyisomeric forms falling within that class (e.g. C₁₋₇ alkyl includesn-propyl and iso-propyl; butyl includes n-, iso-, sec-, and tert-butyl;methoxyphenyl includes ortho-, meta-, and para-methoxyphenyl).

The above exclusion does not pertain to tautomeric forms, for example,keto-, enol-, and enolate-forms, as in, for example, the followingtautomeric pairs: keto/enol (illustrated below), imine/enamine,amide/imino alcohol, amidine/amidine, nitroso/oxime,thioketone/enethiol, N-nitroso/hyroxyazo, and nitro/aci-nitro.

The term “tautomer” or “tautomeric form” refers to structural isomers ofdifferent energies which are interconvertible via a low energy barrier.For example, proton tautomers (also known as prototropic tautomers)include interconversions via migration of a proton, such as keto-enoland imine-enamine isomerizations. Valence tautomers includeinterconversions by reorganization of some of the bonding electrons.

Note that specifically included in the term “isomer” are compounds withone or more isotopic substitutions. For example, H may be in anyisotopic form, including ¹H, ²H (D), and ³H (T); C may be in anyisotopic form, including ¹²C, ¹³C, and ¹⁴C; O may be in any isotopicform, including ¹⁶O and ¹⁸O; and the like.

Examples of isotopes that can be incorporated into compounds of theinvention include isotopes of hydrogen, carbon, nitrogen, oxygen,phosphorous, fluorine, and chlorine, such as, but not limited to ²H(deuterium, D), ³H (tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁸F, ³¹P, ³²P, ³⁵S,³⁶Cl, and ¹²⁵I. Various isotopically labeled compounds of the presentinvention, for example those into which radioactive isotopes such as ³H,¹³C, and ¹⁴C are incorporated. Such isotopically labelled compounds maybe useful in metabolic studies, reaction kinetic studies, detection orimaging techniques, such as positron emission tomography (PET) orsingle-photon emission computed tomography (SPECT) including drug orsubstrate tissue distribution assays, or in radioactive treatment ofpatients. Deuterium labelled or substituted therapeutic compounds of theinvention may have improved DMPK (drug metabolism and pharmacokinetics)properties, relating to distribution, metabolism, and excretion (ADME).Substitution with heavier isotopes such as deuterium may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements. An18F labeled compound may be useful for PET or SPECT studies.Isotopically labeled compounds of this invention and prodrugs thereofcan generally be prepared by carrying out the procedures disclosed inthe schemes or in the examples and preparations described below bysubstituting a readily available isotopically labeled reagent for anon-isotopically labeled reagent. Further, substitution with heavierisotopes, particularly deuterium (i.e., 2H or D) may afford certaintherapeutic advantages resulting from greater metabolic stability, forexample increased in vivo half-life or reduced dosage requirements or animprovement in therapeutic index. It is understood that deuterium inthis context is regarded as a substituent. The concentration of such aheavier isotope, specifically deuterium, may be defined by an isotopicenrichment factor. In the compounds of this invention any atom notspecifically designated as a particular isotope is meant to representany stable isotope of that atom.

Unless otherwise specified, a reference to a particular compoundincludes all such isomeric forms, including (wholly or partially)racemic and other mixtures thereof. Methods for the preparation (e.g.asymmetric synthesis) and separation (e.g. fractional crystallisationand chromatographic means) of such isomeric forms are either known inthe art or are readily obtained by adapting the methods taught herein,or known methods, in a known manner.

Biological Activity

In Vitro Cell Proliferation Assays

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells having receptorproteins to the antibody of the ADC in a cell culture medium; culturingthe cells for a period from about 6 hours to about 5 days; and measuringcell viability. Cell-based in vitro assays are used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of an ADC of the invention.

The in vitro potency of antibody-drug conjugates can be measured by acell proliferation assay. The CellTiter-Glo® Luminescent Cell ViabilityAssay is a commercially available (Promega Corp., Madison, Wis.),homogeneous assay method based on the recombinant expression ofColeoptera luciferase (U.S. Pat. Nos. 5,583,024; 5,674,713 and5,700,670). This cell proliferation assay determines the number ofviable cells in culture based on quantitation of the ATP present, anindicator of metabolically active cells (Crouch et al (1993) J. Immunol.Meth. 160:81-88; U.S. Pat. No. 6,602,677). The CellTiter-Glo® Assay isconducted in 96 well format, making it amenable to automatedhigh-throughput screening (HTS) (Cree et al (1995) AntiCancer Drugs6:398-404). The homogeneous assay procedure involves adding the singlereagent (CellTiter-Glo® Reagent) directly to cells cultured inserum-supplemented medium. Cell washing, removal of medium and multiplepipetting steps are not required. The system detects as few as 15cells/well in a 384-well format in 10 minutes after adding reagent andmixing. The cells may be treated continuously with ADC, or they may betreated and separated from ADC. Generally, cells treated briefly, i.e. 3hours, showed the same potency effects as continuously treated cells.

The homogeneous “add-mix-measure” format results in cell lysis andgeneration of a luminescent signal proportional to the amount of ATPpresent. The amount of ATP is directly proportional to the number ofcells present in culture. The CellTiter-Glo® Assay generates a“glow-type” luminescent signal, produced by the luciferase reaction,which has a half-life generally greater than five hours, depending oncell type and medium used. Viable cells are reflected in relativeluminescence units (RLU). The substrate, Beetle Luciferin, isoxidatively decarboxylated by recombinant firefly luciferase withconcomitant conversion of ATP to AMP and generation of photons.

The in vitro potency of antibody-drug conjugates can also be measured bya cytotoxicity assay. Cultured adherent cells are washed with PBS,detached with trypsin, diluted in complete medium, containing 10% FCS,centrifuged, re-suspended in fresh medium and counted with ahaemocytometer. Suspension cultures are counted directly. Monodispersecell suspensions suitable for counting may require agitation of thesuspension by repeated aspiration to break up cell clumps.

The cell suspension is diluted to the desired seeding density anddispensed (100 μl per well) into black 96 well plates. Plates ofadherent cell lines are incubated overnight to allow adherence.Suspension cell cultures can be used on the day of seeding.

A stock solution (1 ml) of ADC (20 μg/ml) is made in the appropriatecell culture medium. Serial 10-fold dilutions of stock ADC are made in15 ml centrifuge tubes by serially transferring 100 μl to 900 μl of cellculture medium.

Four replicate wells of each ADC dilution (100 μl) are dispensed in96-well black plates, previously plated with cell suspension (100 μl),resulting in a final volume of 200 μl. Control wells receive cellculture medium (100 μl).

If the doubling time of the cell line is greater than 30 hours, ADCincubation is for 5 days, otherwise a four day incubation is done.

At the end of the incubation period, cell viability is assessed with theAlamar blue assay. AlamarBlue (Invitrogen) is dispensed over the wholeplate (20 μl per well) and incubated for 4 hours. Alamar bluefluorescence is measured at excitation 570 nm, emission 585 nm on theVarioskan flash plate reader. Percentage cell survival is calculatedfrom the mean fluorescence in the ADC treated wells compared to the meanfluorescence in the control wells.

Use

The conjugates of the invention may be used to provide a PBD compound ata target location.

The target location is preferably a proliferative cell population. Theantibody is an antibody for an antigen present on a proliferative cellpopulation.

In one embodiment the antigen is absent or present at a reduced level ina non-proliferative cell population compared to the amount of antigenpresent in the proliferative cell population, for example a tumour cellpopulation.

At the target location the linker may be cleaved so as to release acompound RelA. Thus, the conjugate may be used to selectively provide acompound RelA to the target location.

The linker may be cleaved by an enzyme present at the target location.

The target location may be in vitro, in vivo or ex vivo.

The antibody-drug conjugate (ADC) compounds of the invention includethose with utility for anticancer activity. In particular, the compoundsinclude an antibody conjugated, i.e. covalently attached by a linker, toa PBD drug moiety, i.e. toxin. When the drug is not conjugated to anantibody, the PBD drug has a cytotoxic effect. The biological activityof the PBD drug moiety is thus modulated by conjugation to an antibody.The antibody-drug conjugates (ADC) of the invention selectively deliveran effective dose of a cytotoxic agent to tumor tissue whereby greaterselectivity, i.e. a lower efficacious dose, may be achieved.

Thus, in one aspect, the present invention provides a conjugate compoundas described herein for use in therapy.

In a further aspect there is also provides a conjugate compound asdescribed herein for use in the treatment of a proliferative disease. Asecond aspect of the present invention provides the use of a conjugatecompound in the manufacture of a medicament for treating a proliferativedisease.

One of ordinary skill in the art is readily able to determine whether ornot a candidate conjugate treats a proliferative condition for anyparticular cell type. For example, assays which may conveniently be usedto assess the activity offered by a particular compound are described inthe examples below.

The term “proliferative disease” pertains to an unwanted or uncontrolledcellular proliferation of excessive or abnormal cells which isundesired, such as, neoplastic or hyperplastic growth, whether in vitroor in vivo.

Anti-DLK-1 Conjugates

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g.

histocytoma, glioma, astrocyoma, osteoma), cancers (e.g. lung cancer,small cell lung cancer, gastrointestinal cancer, bowel cancer, coloncancer, breast carinoma, ovarian carcinoma, prostate cancer, testicularcancer, liver cancer, kidney cancer, bladder cancer, pancreas cancer,brain cancer, sarcoma, osteosarcoma, Kaposi's sarcoma, melanoma),lymphomas, leukemias, psoriasis, bone diseases, fibroproliferativedisorders (e.g. of connective tissues), and atherosclerosis. Cancers ofparticular interest include, but are not limited to, leukemias andovarian cancers.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Disorders of particular interest include, but are not limited tocancers, including metastatic cancers and metastatic cancer cells, suchas circulating tumour cells, which may be found circulating in bodyfluids such as blood or lymph. Cancers of particular interest include:Hepatocellular carcinoma, hepatoblastoma, non small cell lung cancer,small cell lung cancer, colon cancer, breast cancer, gastric cancer,pancreatic cancer, neuroblastoma, adrenal gland cancer,pheochromocytoma, paraganglioma, thyroid medullary carcinoma, skeletalmuscle cancer, liposarcoma, glioma, Wilms tumor, neuroendocrine tumors,Acute Myeloid Leukemia and Myelodysplastic syndrome.

Other disorders of interest include any condition in which DLK1 isoverexpressed, or wherein DLK1 antagonism will provide a clinicalbenefit. These include immune disorders, cardiovascular disorders,thrombosis, diabetes, immune checkpoint disorders, fibrotic disorders(fibrosis), or proliferative diseases such as cancer, particularlymetastatic cancer.

Anti-KAAG1 Conjugates

Examples of proliferative conditions include, but are not limited to,benign, pre-malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g. ofconnective tissues), and atherosclerosis.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Disorders of particular interest include, but are not limited tocancers, including metastatic cancers and metastatic cancer cells, suchas circulating tumour cells, which may be found circulating in bodyfluids such as blood or lymph. Cancers of particular interest includeovarian, breast, prostate and renal cancer.

Other disorders of interest include any condition in which KAAG1 isoverexpressed, or wherein KAAG1 antagonism will provide a clinicalbenefit. These include immune disorders, cardiovascular disorders,thrombosis, diabetes, immune checkpoint disorders, fibrotic disorders(fibrosis), or proliferative diseases such as cancer, particularlymetastatic cancer.

Anti-Mesothelin Conjugates

Examples of proliferative conditions include, but are not limited to,benign, pre malignant, and malignant cellular proliferation, includingbut not limited to, neoplasms and tumours (e.g. histocytoma, glioma,astrocyoma, osteoma), cancers (e.g. lung cancer, small cell lung cancer,gastrointestinal cancer, bowel cancer, colon cancer, breast carinoma,ovarian carcinoma, prostate cancer, testicular cancer, liver cancer,kidney cancer, bladder cancer, pancreas cancer, brain cancer, sarcoma,osteosarcoma, Kaposi's sarcoma, melanoma), lymphomas, leukemias,psoriasis, bone diseases, fibroproliferative disorders (e.g. ofconnective tissues), and atherosclerosis.

Any type of cell may be treated, including but not limited to, lung,gastrointestinal (including, e.g. bowel, colon), breast (mammary),ovarian, prostate, liver (hepatic), kidney (renal), bladder, pancreas,brain, and skin.

Disorders of particular interest include, but are not limited tocancers, including metastatic cancers and metastatic cancer cells, suchas circulating tumour cells, which may be found circulating in bodyfluids such as blood or lymph. Cancers of particular interest includemesothelioma, lung cancer, ovarian cancer and pancreatic cancer.

Other disorders of interest include any condition in which Mesothelin isoverexpressed, or wherein Mesothelin antagonism will provide a clinicalbenefit. These include immune disorders, cardiovascular disorders,thrombosis, diabetes, immune checkpoint disorders, fibrotic disorders(fibrosis), or proliferative diseases such as cancer, particularlymetastatic cancer.

It is contemplated that the antibody-drug conjugates (ADC) of thepresent invention may be used to treat various diseases or disorders,e.g. characterized by the overexpression of a tumor antigen. Exemplaryconditions or hyperproliferative disorders include benign or malignanttumors; leukemia, haematological, and lymphoid malignancies. Othersinclude neuronal, glial, astrocytal, hypothalamic, glandular,macrophagal, epithelial, stromal, blastocoelic, inflammatory, angiogenicand immunologic, including autoimmune, disorders.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

Autoimmune diseases for which the ADC compounds may be used in treatmentinclude rheumatologic disorders (such as, for example, rheumatoidarthritis, Sjögren's syndrome, scleroderma, lupus such as SLE and lupusnephritis, polymyositis/dermatomyositis, cryoglobulinemia,anti-phospholipid antibody syndrome, and psoriatic arthritis),osteoarthritis, autoimmune gastrointestinal and liver disorders (suchas, for example, inflammatory bowel diseases (e.g. ulcerative colitisand Crohn's disease), autoimmune gastritis and pernicious anemia,autoimmune hepatitis, primary biliary cirrhosis, primary sclerosingcholangitis, and celiac disease), vasculitis (such as, for example,ANCA-associated vasculitis, including Churg-Strauss vasculitis,Wegener's granulomatosis, and polyarteriitis), autoimmune neurologicaldisorders (such as, for example, multiple sclerosis, opsoclonusmyoclonus syndrome, myasthenia gravis, neuromyelitis optica, Parkinson'sdisease, Alzheimer's disease, and autoimmune polyneuropathies), renaldisorders (such as, for example, glomerulonephritis, Goodpasture'ssyndrome, and Berger's disease), autoimmune dermatologic disorders (suchas, for example, psoriasis, urticaria, hives, pemphigus vulgaris,bullous pemphigoid, and cutaneous lupus erythematosus), hematologicdisorders (such as, for example, thrombocytopenic purpura, thromboticthrombocytopenic purpura, post-transfusion purpura, and autoimmunehemolytic anemia), atherosclerosis, uveitis, autoimmune hearing diseases(such as, for example, inner ear disease and hearing loss), Behcet'sdisease, Raynaud's syndrome, organ transplant, and autoimmune endocrinedisorders (such as, for example, diabetic-related autoimmune diseasessuch as insulin-dependent diabetes mellitus (IDDM), Addison's disease,and autoimmune thyroid disease (e.g. Graves' disease and thyroiditis)).More preferred such diseases include, for example, rheumatoid arthritis,ulcerative colitis, ANCA-associated vasculitis, lupus, multiplesclerosis, Sjögren's syndrome, Graves' disease, IDDM, pernicious anemia,thyroiditis, and glomerulonephritis.

Methods of Treatment

The conjugates of the present invention may be used in a method oftherapy. Also provided is a method of treatment, comprisingadministering to a subject in need of treatment atherapeutically-effective amount of a conjugate compound of theinvention. The term “therapeutically effective amount” is an amountsufficient to show benefit to a patient. Such benefit may be at leastamelioration of at least one symptom. The actual amount administered,and rate and time-course of administration, will depend on the natureand severity of what is being treated. Prescription of treatment, e.g.decisions on dosage, is within the responsibility of generalpractitioners and other medical doctors.

A compound of the invention may be administered alone or in combinationwith other treatments, either simultaneously or sequentially dependentupon the condition to be treated. Examples of treatments and therapiesinclude, but are not limited to, chemotherapy (the administration ofactive agents, including, e.g. drugs, such as chemotherapeutics);surgery; and radiation therapy.

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer, regardless of mechanism of action. Classes ofchemotherapeutic agents include, but are not limited to: alkylatingagents, antimetabolites, spindle poison plant alkaloids,cytotoxic/antitumor antibiotics, topoisomerase inhibitors, antibodies,photosensitizers, and kinase inhibitors. Chemotherapeutic agents includecompounds used in “targeted therapy” and conventional chemotherapy.

Examples of chemotherapeutic agents include: erlotinib (TARCEVA®,Genentech/OSI Pharm.), docetaxel (TAXOTERE®, Sanofi-Aventis), 5-FU(fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine (GEMZAR®,Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer), cisplatin(cis-diamine, dichloroplatinum(II), CAS No. 15663-27-1), carboplatin(CAS No. 41575-94-4), paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology,Princeton, N.J.), trastuzumab (HERCEPTIN®, Genentech), temozolomide(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo [4.3.0]nona-2,7,9-triene-9-carboxamide, CAS No. 85622-93-1, TEMODAR®, TEMODAL®,Schering Plough), tamoxifen((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine,NOLVADEX®, ISTUBAL®, VALODEX®), and doxorubicin (ADRIAMYCIN®), Akti-1/2,HPPD, and rapamycin.

More examples of chemotherapeutic agents include: oxaliplatin(ELOXATIN®, Sanofi), bortezomib (VELCADE®, Millennium Pharm.), sutent(SUNITINIB®, SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinibmesylate (GLEEVEC®, Novartis), XL-518 (Mek inhibitor, Exelixis, WO2007/044515), ARRY-886 (Mek inhibitor, AZD6244, Array BioPharma, AstraZeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235(PI3K inhibitor, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK222584 (Novartis), fulvestrant (FASLODEX®, AstraZeneca), leucovorin(folinic acid), rapamycin (sirolimus, RAPAMUNE®, Wyeth), lapatinib(TYKERB®, GSK572016, Glaxo Smith Kline), lonafarnib (SARASAR™, SCH66336, Schering Plough), sorafenib (NEXAVAR®, BAY43-9006, Bayer Labs),gefitinib (IRESSA®, AstraZeneca), irinotecan (CAMPTOSAR®, CPT-11,Pfizer), tipifarnib (ZARNESTRA™, Johnson & Johnson), ABRAXANE™(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, II),vandetanib (rINN, ZD6474, ZACTIMA®, AstraZeneca), chloranmbucil, AG1478,AG1571 (SU 5271; Sugen), temsirolimus (TORISEL®, Wyeth), pazopanib(GlaxoSmithKline), canfosfamide (TELCYTA®, Telik), thiotepa andcyclosphosphamide (CYTOXAN®, NEOSAR®); alkyl sulfonates such asbusulfan, improsulfan and piposulfan; aziridines such as benzodopa,carboquone, meturedopa, and uredopa; ethylenimines and methylamelaminesincluding altretamine, triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analog topotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);dolastatin; duocarmycin (including the synthetic analogs, KW-2189 andCB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin;nitrogen mustards such as chlorambucil, chlornaphazine,chlorophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e.g.calicheamicin, calicheamicin gamma1I, calicheamicin omegal1 (Angew Chem.Intl. Ed. Engl. (1994) 33:183-186); dynemicin, dynemicin A;bisphosphonates, such as clodronate; an esperamicin; as well asneocarzinostatin chromophore and related chromoprotein enediyneantibiotic chromophores), aclacinomysins, actinomycin, authramycin,azaserine, bleomycins, cactinomycin, carabicin, carminomycin,carzinophilin, chromomycinis, dactinomycin, daunorubicin, detorubicin,6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, nemorubicin,marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatrexate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; 6-thioguanine;mercaptopurine; methotrexate; platinum analogs such as cisplatin andcarboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone;vincristine; vinorelbine (NAVELBINE®); novantrone; teniposide;edatrexate; daunomycin; aminopterin; capecitabine (XELODA®, Roche);ibandronate; CPT-11; topoisomerase inhibitor RFS 2000;difluoromethylornithine (DMFO); retinoids such as retinoic acid; andpharmaceutically acceptable salts, acids and derivatives of any of theabove.

Also included in the definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®;tamoxifen citrate), raloxifene, droloxifene, 4-hydroxytamoxifen,trioxifene, keoxifene, LY117018, onapristone, and FARESTON® (toremifinecitrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase,which regulates estrogen production in the adrenal glands, such as, forexample, 4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrolacetate), AROMASIN® (exemestane; Pfizer), formestanie, fadrozole,RIVISOR® (vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX®(anastrozole; AstraZeneca); (iii) anti-androgens such as flutamide,nilutamide, bicalutamide, leuprolide, and goserelin; as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipidkinase inhibitors; (vi) antisense oligonucleotides, particularly thosewhich inhibit expression of genes in signaling pathways implicated inaberrant cell proliferation, for example, PKC-alpha, Raf and H-Ras, suchas oblimersen (GENASENSE®, Genta Inc.); (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN® rIL-2; topoisomerase 1 inhibitorssuch as LURTOTECAN®; ABARELIX® rmRH; (ix) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and pharmaceutically acceptablesalts, acids and derivatives of any of the above.

Also included in the definition of “chemotherapeutic agent” aretherapeutic antibodies such as alemtuzumab (Campath), bevacizumab(AVASTIN®, Genentech); cetuximab (ERBITUX®, lmclone); panitumumab(VECTIBIX®, Amgen), rituximab (RITUXAN®, Genentech/Biogen Idec),ofatumumab (ARZERRA®, GSK), pertuzumab (PERJETA™, OMNITARG™, 2C4,Genentech), trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar,Corixia), and the antibody drug conjugate, gemtuzumab ozogamicin(MYLOTARG®, Wyeth).

Humanized monoclonal antibodies with therapeutic potential aschemotherapeutic agents in combination with the conjugates of theinvention include: alemtuzumab, apolizumab, aselizumab, atlizumab,bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumabmertansine, cedelizumab, certolizumab pegol, cidfusituzumab, cidtuzumab,daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab, felvizumab,fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin, ipilimumab,labetuzumab, lintuzumab, matuzumab, mepolizumab, motavizumab,motovizumab, natalizumab, nimotuzumab, nolovizumab, numavizumab,ocrelizumab, omalizumab, palivizumab, pascolizumab, pecfusituzumab,pectuzumab, pertuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,trastuzumab, tucotuzumab celmoleukin, tucusituzumab, umavizumab,urtoxazumab, and visilizumab.

Pharmaceutical compositions according to the present invention, and foruse in accordance with the present invention, may comprise, in additionto the active ingredient, i.e. a conjugate compound, a pharmaceuticallyacceptable excipient, carrier, buffer, stabiliser or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of the activeingredient. The precise nature of the carrier or other material willdepend on the route of administration, which may be oral, or byinjection, e.g. cutaneous, subcutaneous, or intravenous.

Pharmaceutical compositions for oral administration may be in tablet,capsule, powder or liquid form. A tablet may comprise a solid carrier oran adjuvant. Liquid pharmaceutical compositions generally comprise aliquid carrier such as water, petroleum, animal or vegetable oils,mineral oil or synthetic oil. Physiological saline solution, dextrose orother saccharide solution or glycols such as ethylene glycol, propyleneglycol or polyethylene glycol may be included. A capsule may comprise asolid carrier such a gelatin.

For intravenous, cutaneous or subcutaneous injection, or injection atthe site of affliction, the active ingredient will be in the form of aparenterally acceptable aqueous solution which is pyrogen-free and hassuitable pH, isotonicity and stability. Those of relevant skill in theart are well able to prepare suitable solutions using, for example,isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection,Lactated Ringer's Injection. Preservatives, stabilisers, buffers,antioxidants and/or other additives may be included, as required.

Formulations

While it is possible for the conjugate compound to be used (e.g.,administered) alone, it is often preferable to present it as acomposition or formulation.

In one embodiment, the composition is a pharmaceutical composition(e.g., formulation, preparation, medicament) comprising a conjugatecompound, as described herein, and a pharmaceutically acceptablecarrier, diluent, or excipient.

In one embodiment, the composition is a pharmaceutical compositioncomprising at least one conjugate compound, as described herein,together with one or more other pharmaceutically acceptable ingredientswell known to those skilled in the art, including, but not limited to,pharmaceutically acceptable carriers, diluents, excipients, adjuvants,fillers, buffers, preservatives, anti-oxidants, lubricants, stabilisers,solubilisers, surfactants (e.g., wetting agents), masking agents,colouring agents, flavouring agents, and sweetening agents.

In one embodiment, the composition further comprises other activeagents, for example, other therapeutic or prophylactic agents.

Suitable carriers, diluents, excipients, etc. can be found in standardpharmaceutical texts. See, for example, Handbook of PharmaceuticalAdditives, 2nd Edition (eds. M. Ash and I. Ash), 2001 (SynapseInformation Resources, Inc., Endicott, N.Y., USA), Remington'sPharmaceutical Sciences, 20th edition, pub. Lippincott, Williams &Wilkins, 2000; and Handbook of Pharmaceutical Excipients, 2nd edition,1994.

Another aspect of the present invention pertains to methods of making apharmaceutical composition comprising admixing at least one[¹¹C]-radiolabelled conjugate or conjugate-like compound, as definedherein, together with one or more other pharmaceutically acceptableingredients well known to those skilled in the art, e.g., carriers,diluents, excipients, etc. If formulated as discrete units (e.g.,tablets, etc.), each unit contains a predetermined amount (dosage) ofthe active compound.

The term “pharmaceutically acceptable,” as used herein, pertains tocompounds, ingredients, materials, compositions, dosage forms, etc.,which are, within the scope of sound medical judgment, suitable for usein contact with the tissues of the subject in question (e.g., human)without excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio. Each carrier, diluent, excipient, etc. must also be “acceptable”in the sense of being compatible with the other ingredients of theformulation.

The formulations may be prepared by any methods well known in the art ofpharmacy. Such methods include the step of bringing into association theactive compound with a carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active compound with carriers(e.g., liquid carriers, finely divided solid carrier, etc.), and thenshaping the product, if necessary.

The formulation may be prepared to provide for rapid or slow release;immediate, delayed, timed, or sustained release; or a combinationthereof.

Formulations suitable for parenteral administration (e.g., byinjection), include aqueous or non-aqueous, isotonic, pyrogen-free,sterile liquids (e.g., solutions, suspensions), in which the activeingredient is dissolved, suspended, or otherwise provided (e.g., in aliposome or other microparticulate). Such liquids may additional containother pharmaceutically acceptable ingredients, such as anti-oxidants,buffers, preservatives, stabilisers, bacteriostats, suspending agents,thickening agents, and solutes which render the formulation isotonicwith the blood (or other relevant bodily fluid) of the intendedrecipient. Examples of excipients include, for example, water, alcohols,polyols, glycerol, vegetable oils, and the like. Examples of suitableisotonic carriers for use in such formulations include Sodium ChlorideInjection, Ringer's Solution, or Lactated Ringer's Injection. Typically,the concentration of the active ingredient in the liquid is from about 1ng/ml to about 10 μg/ml, for example from about 10 ng/ml to about 1μg/ml. The formulations may be presented in unit-dose or multi-dosesealed containers, for example, ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example water for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules, and tablets.

Dosage

It will be appreciated by one of skill in the art that appropriatedosages of the conjugate compound, and compositions comprising theconjugate compound, can vary from patient to patient. Determining theoptimal dosage will generally involve the balancing of the level oftherapeutic benefit against any risk or deleterious side effects. Theselected dosage level will depend on a variety of factors including, butnot limited to, the activity of the particular compound, the route ofadministration, the time of administration, the rate of excretion of thecompound, the duration of the treatment, other drugs, compounds, and/ormaterials used in combination, the severity of the condition, and thespecies, sex, age, weight, condition, general health, and prior medicalhistory of the patient. The amount of compound and route ofadministration will ultimately be at the discretion of the physician,veterinarian, or clinician, although generally the dosage will beselected to achieve local concentrations at the site of action whichachieve the desired effect without causing substantial harmful ordeleterious side-effects.

Administration can be effected in one dose, continuously orintermittently (e.g., in divided doses at appropriate intervals)throughout the course of treatment. Methods of determining the mosteffective means and dosage of administration are well known to those ofskill in the art and will vary with the formulation used for therapy,the purpose of the therapy, the target cell(s) being treated, and thesubject being treated. Single or multiple administrations can be carriedout with the dose level and pattern being selected by the treatingphysician, veterinarian, or clinician.

In general, a suitable dose of the active compound is in the range ofabout 100 ng to about 25 mg (more typically about 1 μg to about 10 mg)per kilogram body weight of the subject per day. Where the activecompound is a salt, an ester, an amide, a prodrug, or the like, theamount administered is calculated on the basis of the parent compoundand so the actual weight to be used is increased proportionately.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 100 mg, 3 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 150 mg, 2 timesdaily.

In one embodiment, the active compound is administered to a humanpatient according to the following dosage regime: about 200 mg, 2 timesdaily.

However in one embodiment, the conjugate compound is administered to ahuman patient according to the following dosage regime: about 50 orabout 75 mg, 3 or 4 times daily.

In one embodiment, the conjugate compound is administered to a humanpatient according to the following dosage regime: about 100 or about 125mg, 2 times daily.

The dosage amounts described above may apply to the conjugate (includingthe PBD moiety and the linker to the antibody) or to the effectiveamount of PBD compound provided, for example the amount of compound thatis releasable after cleavage of the linker.

For the prevention or treatment of disease, the appropriate dosage of anADC of the invention will depend on the type of disease to be treated,as defined above, the severity and course of the disease, whether themolecule is administered for preventive or therapeutic purposes,previous therapy, the patient's clinical history and response to theantibody, and the discretion of the attending physician. The molecule issuitably administered to the patient at one time or over a series oftreatments. Depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initialcandidate dosage for administration to the patient, whether, forexample, by one or more separate administrations, or by continuousinfusion. A typical daily dosage might range from about 1 μg/kg to 100mg/kg or more, depending on the factors mentioned above. An exemplarydosage of ADC to be administered to a patient is in the range of about0.1 to about 10 mg/kg of patient weight. For repeated administrationsover several days or longer, depending on the condition, the treatmentis sustained until a desired suppression of disease symptoms occurs. Anexemplary dosing regimen comprises a course of administering an initialloading dose of about 4 mg/kg, followed by additional doses every week,two weeks, or three weeks of an ADC. Other dosage regimens may beuseful. The progress of this therapy is easily monitored by conventionaltechniques and assays.

Treatment

The term “treatment,” as used herein in the context of treating acondition, pertains generally to treatment and therapy, whether of ahuman or an animal (e.g., in veterinary applications), in which somedesired therapeutic effect is achieved, for example, the inhibition ofthe progress of the condition, and includes a reduction in the rate ofprogress, a halt in the rate of progress, regression of the condition,amelioration of the condition, and cure of the condition. Treatment as aprophylactic measure (i.e., prophylaxis, prevention) is also included.

The term “therapeutically-effective amount,” as used herein, pertains tothat amount of an active compound, or a material, composition or dosagefrom comprising an active compound, which is effective for producingsome desired therapeutic effect, commensurate with a reasonablebenefit/risk ratio, when administered in accordance with a desiredtreatment regimen.

Similarly, the term “prophylactically-effective amount,” as used herein,pertains to that amount of an active compound, or a material,composition or dosage from comprising an active compound, which iseffective for producing some desired prophylactic effect, commensuratewith a reasonable benefit/risk ratio, when administered in accordancewith a desired treatment regimen.

Preparation of Drug Conjugates

The antibody drug conjugates of the present invention may be prepared byconjugating the following drug linker:

to the azide-containing antibody by the methods as described in forexample, van Geel, R., et al., Bioconjugate Chemistry, 2015, 26,2233-2242; DOI: 10.1021/acs.bioconjchem.5b00224. Suitable methodsinclude, but are not limited to, copper-free conjugation, in forexample, aqueous conditions with an optional cosolvent selected fromDMF, DMSO and DMA.

The drug linker may be synthesised in accordance with the examples, withappropriate modifications, for example, referring to WO 2016/053107 forsynthesis of the linker and the following documents for the PBD dimer,for example: WO 2011/130598, WO2013/055987, WO2014/057074.

The Subject/Patient

The subject/patient may be an animal, mammal, a placental mammal, amarsupial (e.g., kangaroo, wombat), a monotreme (e.g., duckbilledplatypus), a rodent (e.g., a guinea pig, a hamster, a rat, a mouse),murine (e.g., a mouse), a lagomorph (e.g., a rabbit), avian (e.g., abird), canine (e.g., a dog), feline (e.g., a cat), equine (e.g., ahorse), porcine (e.g., a pig), ovine (e.g., a sheep), bovine (e.g., acow), a primate, simian (e.g., a monkey or ape), a monkey (e.g.,marmoset, baboon), an ape (e.g., gorilla, chimpanzee, orangutang,gibbon), or a human.

Furthermore, the subject/patient may be any of its forms of development,for example, a foetus. In one preferred embodiment, the subject/patientis a human.

STATEMENTS OF INVENTION

The following numbered statements describe some specifically envisionedcombinations of the present invention.

Anti-DLK1 Conjugates 1. A conjugate of formula (I):Ab-(DL)_(p)  (I)

wherein:

Ab is an antibody that binds to DLK1;

DL is

wherein:

X is selected from the group comprising: a single bond, —CH₂— and—C₂H₄—;

n is from 1 to 8;

m is 0 or 1;

R⁷ is either methyl or phenyl;

when there is a double bond between C2 and C3, R² is selected the groupconsisting of:

(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁₋₅ saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2 and C3, R² is

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

when there is a double bond between C2′ and C3′, R¹² is selected thegroup consisting of:

(iia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃-7 heterocyclyl and bis-oxy-C₁₋₃alkylene;

(iib) C₁₋₅ saturated aliphatic alkyl;

(iic) C₃₋₆ saturated cycloalkyl;

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(35a) and R^(35b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2′ and C3′, R¹² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester; and p is from 1 to 8.

2. The conjugate according to statement 1, wherein X is a single bond.

3. The conjugate according to statement 1, wherein X is —CH₂—.

4. The conjugate according to statement 1, wherein X is —C₂H₄—.

5. The conjugate according to any one of statements 1 to 4, wherein n is1 to 4.

6. The conjugate according to statement 5, wherein n is 1.

7. The conjugate according to statement 5, wherein n is 2.

8. The conjugate according to statement 5, wherein n is 4.

9. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C5-7 aryl group.

10. A compound according to statement 9, wherein R² is phenyl.

11. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₈₋₁₀ aryl group.

12. A compound according to any one of statements 9 to 11, wherein R²bears one to three substituent groups.

13. A compound according to any one of statements 9 to 12, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

14. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₁₋₅ saturated aliphaticalkyl group.

15. A compound according to statement 14, wherein R² is methyl, ethyl orpropyl.

16. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₃₋₆ saturatedcycloalkyl group.

17. A compound according to statement 16, wherein R² is cyclopropyl.

18. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

19. A compound according to statement 18, wherein the total number ofcarbon atoms in the R² group is no more than 4.

20. A compound according to statement 19, wherein the total number ofcarbon atoms in the R² group is no more than 3.

21. A compound according to any one of statements 18 to 20, wherein oneof R²¹, R²² and R²³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

22. A compound according to any one of statements 18 to 20, wherein twoof R²¹, R²² and R²³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

23. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C₂ and C3, and R² is a group of formula:

24. A compound according to statement 23, wherein R² is the group:

25. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

26. A compound according to statement 25, wherein R²⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

27. A compound according to statement 26, wherein R²⁴ is selected from Hand methyl.

28. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both H.

29. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both methyl.

30. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

one of R^(26a) and R^(26b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

31. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C5-7 aryl group.

32. A compound according to statement 31, wherein R¹² is phenyl.

33. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₈₋₁₀ aryl group.

34. A compound according to any one of statements 31 to 33, wherein R¹²bears one to three substituent groups.

35. A compound according to any one of statements 31 to 34, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

36. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₁₋₅ saturatedaliphatic alkyl group.

37. A compound according to statement 36, wherein R¹² is methyl, ethylor propyl.

38. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₃₋₆ saturatedcycloalkyl group.

39. A compound according to statement 38, wherein R¹² is cyclopropyl.

40. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

41. A compound according to statement 40, wherein the total number ofcarbon atoms in the R¹² group is no more than 4.

42. A compound according to statement 41, wherein the total number ofcarbon atoms in the R¹² group is no more than 3.

43. A compound according to any one of statements 40 to 42, wherein oneof R³¹, R³² and R³³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

44. A compound according to any one of statements 40 to 42, wherein twoof R³¹, R³² and R³³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

45. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

46. A compound according to statement 45, wherein R¹² is the group:

47. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

48. A compound according to statement 47, wherein R³⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

49. A compound according to statement 48, wherein R³⁴ is selected from Hand methyl.

50. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both H.

51. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both methyl.

52. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

one of R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

53. A conjugate according to statement 1, wherein DL is:

54. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.7.

55. The conjugate according to any one of statements 1 to 54 wherein theantibody comprises a VH domain comprising a VH CDR2 with the amino acidsequence of SEQ ID NO.6, and/or a VH CDR1 with the amino acid sequenceof SEQ ID NO.5.

56. The conjugate according to any one of statements 1 to 55 wherein theantibody comprises a VH domain comprising a VH CDR3 with the amino acidsequence of SEQ ID NO.7., a VH CDR2 with the amino acid sequence of SEQID NO.6, and a VH CDR1 with the amino acid sequence of SEQ ID NO.5.

57. The conjugate according to any one of statements 1 to 56 wherein theantibody comprises a VH domain having the sequence of SEQ ID NO.1.

58. The conjugate according to any one of statements 1 to 57 wherein theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.10.

59. The conjugate according to any one of statements 1 to 58 wherein theantibody comprises a VL domain comprising a VL CDR2 with the amino acidsequence of SEQ ID NO.9, and/or a VL CDR1 with the amino acid sequenceof SEQ ID NO.8.

60. The conjugate according to any one of statements 1 to 59 wherein theantibody comprises a VL domain comprising a VL CDR3 with the amino acidsequence of SEQ ID NO.10, a VL CDR2 with the amino acid sequence of SEQID NO.9, and a VL CDR1 with the amino acid sequence of SEQ ID NO.8.

61. The conjugate according to any one of statements 1 to 60 wherein theantibody comprises a VL domain having the sequence of SEQ ID NO. 2.

62. The conjugate according to any one of statements 1 to 61 wherein theantibody in an intact antibody.

63. The conjugate according to any one of statements 1 to 62, whereinthe antibody comprises a heavy chain having the sequence of SEQ ID NO.3, or a heavy chain having the sequence of SEQ ID NO. 11.

64. The conjugate according to any one of statements 1 to 63, whereinthe antibody comprises a light chain having the sequence of SEQ ID NO.4.

65. The conjugate according to any one of statements 1 to 64 wherein theantibody is humanised, deimmunised or resurfaced.

66. The conjugate according to any one of statements 1 to 65, whereinthere are no unconujated azide groups on the antibody.

67. The conjugate according to any one of statements 1 to 66, wherein pis 1, 2, 3, or 4.

68. A composition comprising a mixture of the antibody-drug conjugatecompounds as defined in any one of statements 1 to 67, wherein theaverage drug loading per antibody in the mixture of antibody-drugconjugate compounds is about 1 to about 4.

69. The conjugate according to any one of statements 1 to 67, for use intherapy.

70. The conjugate according to any one of statements 1 to 67, for use inthe treatment of a proliferative disease in a subject.

71. The conjugate according to statement 70, wherein the disease iscancer.

72. The conjugate according to statement 71, wherein the cancer is acancer selected from the group consisting of: Hepatocellular carcinoma,hepatoblastoma, non small cell lung cancer, small cell lung cancer,colon cancer, breast cancer, gastric cancer, pancreatic cancer,neuroblastoma, adrenal gland cancer, pheochromocytoma, paraganglioma,thyroid medullary carcinoma, skeletal muscle cancer, liposarcoma,glioma, Wilms tumor, neuroendocrine tumors, Acute Myeloid Leukemia, andMyelodysplastic syndrome.

73. A pharmaceutical composition comprising the conjugate of any one ofstatements 1 to 67 and a pharmaceutically acceptable diluent, carrier orexcipient.

74. The pharmaceutical composition of statement 73 further comprising atherapeutically effective amount of a chemotherapeutic agent.

75. Use of a conjugate according to any one of statements 1 to 67 in thepreparation of a medicament for use in the treatment of a proliferativedisease in a subject.

76. A method of treating cancer comprising administering to a patientthe pharmaceutical composition of statements 74.

77. The method of statement 76 wherein the patient is administered achemotherapeutic agent, in combination with the conjugate.

Anti-KAAG1 conjugates

1. A conjugate of formula (I):Ab-(DL)_(p)  (I)

wherein:

Ab is an antibody that binds to KAAG1;

DL is

wherein:

X is selected from the group comprising: a single bond, —CH₂— and—C₂H₄—;

n is from 1 to 8;

m is 0 or 1;

R⁷ is either methyl or phenyl;

when there is a double bond between C2 and C3, R² is selected the groupconsisting of:

(ia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁₋₅ saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2 and C3, R² is

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester;

when there is a double bond between C2′ and C3′, R¹² is selected thegroup consisting of:

(iia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(iib) C₁₋₅ saturated aliphatic alkyl;

(iic) C₃₋₆ saturated cycloalkyl;

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(35a) and R^(35b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2′ and C3′, R¹² is

where R^(36a) and R^(36b) are independently selected from H, F, C1-4saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C1-4 alkyl ester;

and p is from 1 to 8.

2. The conjugate according to statement 1, wherein X is a single bond.

3. The conjugate according to statement 1, wherein X is —CH₂—.

4. The conjugate according to statement 1, wherein X is —C₂H₄—.

5. The conjugate according to any one of statements 1 to 4, wherein n is1 to 4.

6. The conjugate according to statement 5, wherein n is 1.

7. The conjugate according to statement 5, wherein n is 2.

8. The conjugate according to statement 5, wherein n is 4.

9. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C₂ and C3, and R² is a C₅₋₇ aryl group.

10. A compound according to statement 9, wherein R² is phenyl.

11. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₅₋₁₀ aryl group.

12. A compound according to any one of statements 9 to 11, wherein R²bears one to three substituent groups.

13. A compound according to any one of statements 9 to 12, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

14. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₁₋₅ saturated aliphaticalkyl group.

15. A compound according to statement 14, wherein R² is methyl, ethyl orpropyl.

16. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C3-6 saturatedcycloalkyl group.

17. A compound according to statement 16, wherein R² is cyclopropyl.

18. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

19. A compound according to statement 18, wherein the total number ofcarbon atoms in the R² group is no more than 4.

20. A compound according to statement 19, wherein the total number ofcarbon atoms in the R² group is no more than 3.

21. A compound according to any one of statements 18 to 20, wherein oneof R²¹, R²² and R²³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

22. A compound according to any one of statements 18 to 20, wherein twoof R²¹, R²² and R²³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C2-3 alkynyl and cyclopropyl.

23. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

24. A compound according to statement 23, wherein R² is the group:

25. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

26. A compound according to statement 25, wherein R²⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

27. A compound according to statement 26, wherein R²⁴ is selected from Hand methyl.

28. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both H.

29. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both methyl.

30. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

one of R^(26a) and R^(26b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

31. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₅₋₇ aryl group.

32. A compound according to statement 31, wherein R¹² is phenyl.

33. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₈₋₁₀ aryl group.

34. A compound according to any one of statements 31 to 33, wherein R¹²bears one to three substituent groups.

35. A compound according to any one of statements 31 to 34, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

36. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₁₋₅ saturatedaliphatic alkyl group.

37. A compound according to statement 36, wherein R¹² is methyl, ethylor propyl.

38. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₃₋₆ saturatedcycloalkyl group.

39. A compound according to statement 38, wherein R¹² is cyclopropyl.

40. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

41. A compound according to statement 40, wherein the total number ofcarbon atoms in the R¹² group is no more than 4.

42. A compound according to statement 41, wherein the total number ofcarbon atoms in the R¹² group is no more than 3.

43. A compound according to any one of statements 40 to 42, wherein oneof R³¹, R³² and R³³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

44. A compound according to any one of statements 40 to 42, wherein twoof R³¹, R³² and R³³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

45. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

46. A compound according to statement 45, wherein R¹² is the group:

47. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

48. A compound according to statement 47, wherein R³⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

49. A compound according to statement 48, wherein R³⁴ is selected from Hand methyl.

50. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both H.

51. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both methyl.

52. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

one of R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

53. A conjugate according to statement 1, wherein DL is:

54. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.107.

55. The conjugate according to any one of statements 1 to 54 wherein theantibody comprises a VH domain comprising a VH CDR2 with the amino acidsequence of SEQ ID NO.106, and/or a VH CDR1 with the amino acid sequenceof SEQ ID NO.105.

56. The conjugate according to any one of statements 1 to 55 wherein theantibody comprises a VH domain comprising a VH CDR3 with the amino acidsequence of SEQ ID

NO.107, a VH CDR2 with the amino acid sequence of SEQ ID NO.106, and aVH CDR1 with the amino acid sequence of SEQ ID NO.105.

57. The conjugate according to any one of statements 1 to 56 wherein theantibody comprises a VH domain having the sequence of SEQ ID NO.101.

58. The conjugate according to any one of statements 1 to 57 wherein theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.110.

59. The conjugate according to any one of statements 1 to 58 wherein theantibody comprises a VL domain comprising a VL CDR2 with the amino acidsequence of SEQ ID NO.109, and/or a VL CDR1 with the amino acid sequenceof SEQ ID NO.108.

60. The conjugate according to any one of statements 1 to 59 wherein theantibody comprises a VL domain comprising a VL CDR3 with the amino acidsequence of SEQ ID NO.110, a VL CDR2 with the amino acid sequence of SEQID NO.109, and a VL CDR1 with the amino acid sequence of SEQ ID NO.108.

61. The conjugate according to any one of statements 1 to 60 wherein theantibody comprises a VL domain having the sequence of SEQ ID NO.102.

62. The conjugate according to any one of statements 1 to 60 wherein theantibody comprises a VL domain having the sequence of SEQ ID NO.113. 63.The conjugate according to any one of statements 1 to 60 wherein theantibody comprises a VL domain having the sequence of SEQ ID NO. 115.

64. The conjugate according to any one of statements 1 to 63 wherein theantibody in an intact antibody.

65. The conjugate according to any one of statements 1 to 64, whereinthe antibody comprises a heavy chain having the sequence of SEQ IDNO.103, or a heavy chain having the sequence of SEQ ID NO.111.

66. The conjugate according to any one of statements 1 to 65, whereinthe antibody comprises a light chain having the sequence of SEQ IDNO.104.

67. The conjugate according to any one of statements 1 to 65, whereinthe antibody comprises a light chain having the sequence of SEQ IDNO.114.

68. The conjugate according to any one of statements 1 to 65, whereinthe antibody comprises a light chain having the sequence of SEQ IDNO.116.

69. The conjugate according to any one of statements 1 to 68 wherein theantibody is humanised, deimmunised or resurfaced.

70. The conjugate according to any one of statements 1 to 69, whereinthere are no unconujated azide groups on the antibody.

71. The conjugate according to any one of statements 1 to 70, wherein pis 1, 2, 3, or 4.

72. A composition comprising a mixture of the antibody-drug conjugatecompounds as defined in any one of statements 1 to 71, wherein theaverage drug loading per antibody in the mixture of antibody-drugconjugate compounds is about 1 to about 4.

73. The conjugate according to any one of statements 1 to 71, for use intherapy.

74. The conjugate according to any one of statements 1 to 71, for use inthe treatment of a proliferative disease in a subject.

75. The conjugate according to statement 74, wherein the disease iscancer.

76. The conjugate according to statement 75, wherein the cancer is acancer selected from the group consisting of: ovarian, breast, prostateand renal cancer.

77. A pharmaceutical composition comprising the conjugate of any one ofstatements 1 to 71 and a pharmaceutically acceptable diluent, carrier orexcipient.

78. The pharmaceutical composition of statement 77 further comprising atherapeutically effective amount of a chemotherapeutic agent.

79. Use of a conjugate according to any one of statements 1 to 71 in thepreparation of a medicament for use in the treatment of a proliferativedisease in a subject.

80. A method of treating cancer comprising administering to a patientthe pharmaceutical composition of statements 78.

81. The method of statement 80 wherein the patient is administered achemotherapeutic agent, in combination with the conjugate.

Anti-Mesothelin conjugates

1. A conjugate of formula (I):Ab-(DL)_(p)  (I)

wherein:

Ab is an antibody that binds to Mesothelin;

DL is

wherein:

X is selected from the group comprising: a single bond, —CH₂— and—C₂H₄—;

n is from 1 to 8;

m is 0 or 1;

R⁷ is either methyl or phenyl;

when there is a double bond between C2 and C3, R² is selected the groupconsisting of:

(ia) C5-10 aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(ib) C₁₋₅ saturated aliphatic alkyl;

(ic) C₃₋₆ saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R^(25a) and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2 and C3, R² is

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(26a) and R^(26b) is H, the other isselected from nitrile and a C1-4 alkyl ester;

when there is a double bond between C2′ and C3′, R¹² is selected thegroup consisting of:

(iia) C₅₋₁₀ aryl group, optionally substituted by one or moresubstituents selected from the group comprising: halo, nitro, cyano,ether, carboxy, ester, C1-7 alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃alkylene;

(iib) C₁₋₅ saturated aliphatic alkyl;

(iic) C₃₋₆ saturated cycloalkyl;

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(35a) and R^(35b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy; pyridyl; and thiophenyl; and

where R²⁴ is selected from: H; C₁₋₃ saturated alkyl; C₂₋₃ alkenyl; C₂₋₃alkynyl; cyclopropyl; phenyl, which phenyl is optionally substituted bya group selected from halo, methyl, methoxy; pyridyl; and thiophenyl;

when there is a single bond between C2′ and C3′, R¹² is

where R^(36a) and R^(36b) are independently selected from H, F, C1-4saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C1-4 alkyl ester;

and p is from 1 to 8.

2. The conjugate according to statement 1, wherein X is a single bond.

3. The conjugate according to statement 1, wherein X is —CH₂—.

4. The conjugate according to statement 1, wherein X is —C₂H₄—.

5. The conjugate according to any one of statements 1 to 4, wherein n is1 to 4.

6. The conjugate according to statement 5, wherein n is 1.

7. The conjugate according to statement 5, wherein n is 2.

8. The conjugate according to statement 5, wherein n is 4.

9. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₅₋₇ aryl group.

10. A compound according to statement 9, wherein R² is phenyl.

11. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₈₋₁₀ aryl group.

12. A compound according to any one of statements 9 to 11, wherein R²bears one to three substituent groups.

13. A compound according to any one of statements 9 to 12, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

14. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C₁₋₅ saturated aliphaticalkyl group.

15. A compound according to statement 14, wherein R² is methyl, ethyl orpropyl.

16. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a C3-6 saturatedcycloalkyl group.

17. A compound according to statement 16, wherein R² is cyclopropyl.

18. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

19. A compound according to statement 18, wherein the total number ofcarbon atoms in the R² group is no more than 4.

20. A compound according to statement 19, wherein the total number ofcarbon atoms in the R² group is no more than 3.

21. A compound according to any one of statements 18 to 20, wherein oneof R²¹, R²² and R²³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

22. A compound according to any one of statements 18 to 20, wherein twoof R²¹, R²² and R²³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C2-3 alkynyl and cyclopropyl.

23. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

24. A compound according to statement 23, wherein R² is the group:

25. A compound according to any one of statements 1 to 8, wherein thereis a double bond between C2 and C3, and R² is a group of formula:

26. A compound according to statement 25, wherein R²⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

27. A compound according to statement 26, wherein R²⁴ is selected from Hand methyl.

28. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both H.

29. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

and R^(26a) and R^(26b) are both methyl.

30. A compound according to any one of statements 1 to 8, wherein thereis a single bond between C2 and C3, R² is

one of R^(26a) and R^(26b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

31. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₅₋₇ aryl group.

32. A compound according to statement 31, wherein R¹² is phenyl.

33. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₈₋₁₀ aryl group.

34. A compound according to any one of statements 31 to 33, wherein R¹²bears one to three substituent groups.

35. A compound according to any one of statements 31 to 34, wherein thesubstituents are selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl.

36. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₁₋₅ saturatedaliphatic alkyl group.

37. A compound according to statement 36, wherein R¹² is methyl, ethylor propyl.

38. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a C₃₋₆ saturatedcycloalkyl group.

39. A compound according to statement 38, wherein R¹² is cyclopropyl.

40. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

41. A compound according to statement 40, wherein the total number ofcarbon atoms in the R¹² group is no more than 4.

42. A compound according to statement 41, wherein the total number ofcarbon atoms in the R¹² group is no more than 3.

43. A compound according to any one of statements 40 to 42, wherein oneof R³¹, R³² and R³³ is H, with the other two groups being selected fromH, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

44. A compound according to any one of statements 40 to 42, wherein twoof R³¹, R³² and R³³ are H, with the other group being selected from H,C₁₋₃ saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl.

45. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

46. A compound according to statement 45, wherein R¹² is the group:

47. A compound according to any one of statements 1 to 30, wherein thereis a double bond between C2′ and C3′, and R¹² is a group of formula:

48. A compound according to statement 47, wherein R³⁴ is selected fromH, methyl, ethyl, ethenyl and ethynyl.

49. A compound according to statement 48, wherein R³⁴ is selected from Hand methyl.

50. A compound according to any one of statements 1 to 30, wherein thereis a single

bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both H.

51. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

and R^(36a) and R^(36b) are both methyl.

52. A compound according to any one of statements 1 to 30, wherein thereis a single bond between C2′ and C3′, R¹² is

one of R^(36a) and R^(36b) is H, and the other is selected from C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted.

53. A conjugate according to statement 1, wherein DL is:

54. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.207.

55. The conjugate according to any one of statements 1 to 54 wherein theantibody comprises a VH domain comprising a VH CDR2 with the amino acidsequence of SEQ ID NO.206, and/or a VH CDR1 with the amino acid sequenceof SEQ ID NO.205.

56. The conjugate according to any one of statements 1 to 55 wherein theantibody comprises a VH domain comprising a VH CDR3 with the amino acidsequence of SEQ ID

NO.207, a VH CDR2 with the amino acid sequence of SEQ ID NO.206, and aVH CDR1 with the amino acid sequence of SEQ ID NO.205.

57. The conjugate according to any one of statements 1 to 56 wherein theantibody comprises a VH domain having the sequence of SEQ ID NO.201.

58. The conjugate according to any one of statements 1 to 57 wherein theantibody comprises a VL domain having a VL CDR3 with the amino acidsequence of SEQ ID NO.210.

59. The conjugate according to any one of statements 1 to 58 wherein theantibody comprises a VL domain comprising a VL CDR2 with the amino acidsequence of SEQ ID NO.209, and/or a VL CDR1 with the amino acid sequenceof SEQ ID NO.208.

60. The conjugate according to any one of statements 1 to 59 wherein theantibody comprises a VL domain comprising a VL CDR3 with the amino acidsequence of SEQ ID NO.210, a VL CDR2 with the amino acid sequence of SEQID NO.209, and a VL CDR1 with the amino acid sequence of SEQ ID NO.208.

61. The conjugate according to any one of statements 1 to 60 wherein theantibody comprises a VL domain having the sequence of SEQ ID NO. 202.

62. The conjugate according to any one of statements 1 to 61 wherein theantibody in an intact antibody.

63. The conjugate according to any one of statements 1 to 62, whereinthe antibody comprises a heavy chain having the sequence of SEQ IDNO.203, or a heavy chain having the sequence of SEQ ID NO. 211.

64. The conjugate according to any one of statements 1 to 63, whereinthe antibody comprises a light chain having the sequence of SEQ IDNO.204.

65. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.218.

66. The conjugate according to any one of statements 1 to 53 or 65wherein the antibody comprises a VH domain comprising a VH CDR2 with theamino acid sequence of SEQ ID NO.217, and/or a VH CDR1 with the aminoacid sequence of SEQ ID NO.216.

67. The conjugate according to any one of statements 1 to 53 or 65 to 66wherein the antibody comprises a VH domain comprising a VH CDR3 with theamino acid sequence of SEQ ID NO.218., a VH CDR2 with the amino acidsequence of SEQ ID NO.217, and a VH CDR1 with the amino acid sequence ofSEQ ID NO.216.

68. The conjugate according to any one of statements 1 to 53 or 65 to 67wherein the antibody comprises a VH domain having the sequence of SEQ IDNO.212.

69. The conjugate according to any one of statements 1 to 53 or 65 to 68wherein the antibody comprises a VL domain having a VL CDR3 with theamino acid sequence of SEQ ID NO.221.

70. The conjugate according to any one of statements 1 to 53 or 65 to 69wherein the antibody comprises a VL domain comprising a VL CDR2 with theamino acid sequence of SEQ ID NO.220, and/or a VL CDR1 with the aminoacid sequence of SEQ ID NO.219.

71. The conjugate according to any one of statements 1 to 53 or 65 to 70wherein the antibody comprises a VL domain comprising a VL CDR3 with theamino acid sequence of SEQ ID NO.221., a VL CDR2 with the amino acidsequence of SEQ ID NO.220, and a VL CDR1 with the amino acid sequence ofSEQ ID NO.219.

72. The conjugate according to any one of statements 1 to 53 or 65 to 71wherein the antibody comprises a VL domain having the sequence of SEQ IDNO. 213.

73. The conjugate according to any one of statements 1 to 53 or 65 to 72wherein the antibody in an intact antibody.

74. The conjugate according to any one of statements 1 to 53 or 65 to73, wherein the antibody comprises a heavy chain having the sequence ofSEQ ID NO. 214, or a heavy chain having the sequence of SEQ ID NO. 222.

75. The conjugate according to any one of statements 1 to 53 or 65 to74, wherein the antibody comprises a light chain having the sequence ofSEQ ID NO. 215.

76. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.229.

77. The conjugate according to any one of statements 1 to 53 or 76wherein the antibody comprises a VH domain comprising a VH CDR2 with theamino acid sequence of SEQ ID NO.228, and/or a VH CDR1 with the aminoacid sequence of SEQ ID NO.227.

78. The conjugate according to any one of statements 1 to 53 or 76 to 77wherein the antibody comprises a VH domain comprising a VH CDR3 with theamino acid sequence of SEQ ID NO.228., a VH CDR2 with the amino acidsequence of SEQ ID NO.227, and a VH CDR1 with the amino acid sequence ofSEQ ID NO.226.

79. The conjugate according to any one of statements 1 to 53 or 76 to 78wherein the antibody comprises a VH domain having the sequence of SEQ IDNO.223.

80. The conjugate according to any one of statements 1 to 53 or 76 to 79wherein the antibody comprises a VL domain having a VL CDR3 with theamino acid sequence of SEQ ID NO.232.

81. The conjugate according to any one of statements 1 to 53 or 76 to 80wherein the antibody comprises a VL domain comprising a VL CDR2 with theamino acid sequence of SEQ ID NO.231, and/or a VL CDR1 with the aminoacid sequence of SEQ ID NO.230.

82. The conjugate according to any one of statements 1 to 53 or 76 to 81wherein the antibody comprises a VL domain comprising a VL CDR3 with theamino acid sequence of SEQ ID NO.232., a VL CDR2 with the amino acidsequence of SEQ ID NO.231, and a VL CDR1 with the amino acid sequence ofSEQ ID NO.230.

83. The conjugate according to any one of statements 1 to 53 or 76 to 82wherein the antibody comprises a VL domain having the sequence of SEQ IDNO. 224.

84. The conjugate according to any one of statements 1 to 53 or 76 to 83wherein the antibody in an intact antibody.

85. The conjugate according to any one of statements 1 to 53 or 76 to84, wherein the antibody comprises a heavy chain having the sequence ofSEQ ID NO.225, or a heavy chain having the sequence of SEQ ID NO.233.

86. The conjugate according to any one of statements 1 to 53 or 76 to85, wherein the antibody comprises a light chain having the sequence ofSEQ ID NO.226.

87. The conjugate according to any one of statements 1 to 53 wherein theantibody comprises a VH domain having a VH CDR3 with the amino acidsequence of SEQ ID NO.240.

88. The conjugate according to any one of statements 1 to 53 or 87wherein the antibody comprises a VH domain comprising a VH CDR2 with theamino acid sequence of SEQ ID NO.239, and/or a VH CDR1 with the aminoacid sequence of SEQ ID NO.238.

89. The conjugate according to any one of statements 1 to 53 or 87 to 88wherein the antibody comprises a VH domain comprising a VH CDR3 with theamino acid sequence of SEQ ID NO.240, a VH CDR2 with the amino acidsequence of SEQ ID NO.239, and a VH CDR1 with the amino acid sequence ofSEQ ID NO.238.

90. The conjugate according to any one of statements 1 to 53 or 87 to 89wherein the antibody comprises a VH domain having the sequence of SEQ IDNO.234. 91. The conjugate according to any one of statements 1 to 53 or87 to 90 wherein the antibody comprises a VL domain having a VL CDR3with the amino acid sequence of SEQ ID NO.243.

92. The conjugate according to any one of statements 1 to 53 or 87 to 91wherein the antibody comprises a VL domain comprising a VL CDR2 with theamino acid sequence of SEQ ID NO.242, and/or a VL CDR1 with the aminoacid sequence of SEQ ID NO.241.

93. The conjugate according to any one of statements 1 to 53 or 87 to 92wherein the antibody comprises a VL domain comprising a VL CDR3 with theamino acid sequence of

SEQ ID NO.243., a VL CDR2 with the amino acid sequence of SEQ ID NO.242,and a VL CDR1 with the amino acid sequence of SEQ ID NO.241.

94. The conjugate according to any one of statements 1 to 53 or 87 to 93wherein the antibody comprises a VL domain having the sequence of SEQ IDNO.235.

95. The conjugate according to any one of statements 1 to 53 or 87 to 94wherein the antibody in an intact antibody.

96. The conjugate according to any one of statements 1 to 53 or 87 to95, wherein the antibody comprises a heavy chain having the sequence ofSEQ ID NO.236, or a heavy chain having the sequence of SEQ ID NO.244.

97. The conjugate according to any one of statements 1 to 53 or 87 to96, wherein the antibody comprises a light chain having the sequence ofSEQ ID NO.237.

98. The conjugate according to any one of statements 1 to 97 wherein theantibody is humanised, deimmunised or resurfaced.

99. The conjugate according to any one of statements 1 to 98, whereinthere are no unconujated azide groups on the antibody.

100. The conjugate according to any one of statements 1 to 99, wherein pis 1, 2, 3, or 4.

101. A composition comprising a mixture of the antibody-drug conjugatecompounds as defined in any one of statements 1 to 100, wherein theaverage drug loading per antibody in the mixture of antibody-drugconjugate compounds is about 1 to about 4.

102. The conjugate according to any one of statements 1 to 100, for usein therapy.

103. The conjugate according to any one of statements 1 to 100, for usein the treatment of a proliferative disease in a subject.

104. The conjugate according to statement 103, wherein the disease iscancer.

105. The conjugate according to statement 104, wherein the cancer is acancer selected from the group consisting of: mesothelioma, lung cancer,ovarian cancer and pancreatic cancer.

106. A pharmaceutical composition comprising the conjugate of any one ofstatements 1 to 100 and a pharmaceutically acceptable diluent, carrieror excipient.

107. The pharmaceutical composition of statement 106 further comprisinga therapeutically effective amount of a chemotherapeutic agent.

108. Use of a conjugate according to any one of statements 1 to 100 inthe preparation of a medicament for use in the treatment of aproliferative disease in a subject.

109. A method of treating cancer comprising administering to a patientthe pharmaceutical composition of statement 101.

110. The method of statement 109 wherein the patient is administered achemotherapeutic agent, in combination with the conjugate.

EXAMPLES

Overview

The ADCs disclosed herein are synthesised in a two-stage process. Thefirst stage—glycan remodelling—trims the native N-linked glycan back tothe core GlcNAc and then adds an azido modified GalNAc to form anantibody-GalNAc-N₃ intermediate. This is purified by Protein Achromatography and then conjugated to the drug linker in the secondstage.

These stages are described below the following description of how thedrug-linker is synthesised.

Synthesis of Intermediate 3

A solution of BCN alcohol (0.384 g, 2.55 mmole) in MeCN (25 mL) under aN₂ atmosphere was cooled to 0° C., and chlorosulfonyl isocyanate wasadded (CSI) was added dropwise (0.255 mL, 415 mg, 2.93 mmole, 1.15equiv.). After stirring for 15 minutes, Et₃N was added dropwise (1.42mL, 1.03 g, 10.2 mmole, 4 equiv.) and stirring was continued for another10 minutes. Next, a solution of 2-(2-(2-aminoethoxy)ethoxy)acetic acid(1.0 g, 6.1 mmole, 2.4 equiv.) in H₂O (5 mL) was added and the reactionmixture was stirred to room temperature for 2 h. After this time, CHCl₃(50 mL) and H₂O (100 mL) were added, and the layers were separated. Tothe aqueous layer in a separatory funnel was added CH₂Cl₂ (100 mL) andthe pH was adjusted to 4 with 1 N HCl, before separation of layers. Thewater layer was extracted twice with CH₂Cl₂ (2×100 mL), the organiclayers were combined and dried (Na₂SO₄), filtered and concentrated. Theresidue was purified by flask column chromatography on silica, elutionwith CH₂Cl₂ to 20% MeOH in CH₂Cl₂. Yield 0.42 g (1.0 mmole, 39%) of 3 asa colorless sticky wax.

Synthesis of Drug Linker

Compound 1 can be synthesised as described in WO2014/057074—see compound22.

(a) Palladium tetrakistriphenylphosphine (Pd(PPh₃)₄, 4.8 mg, 4.15 μmol)is weighed and put under an inert atmosphere. A solution of pyrrolidine(5.0 μL, 4.3 mg, 60 μmol) in DCM (1 mL) is degassed by bubbling N2through the solution. A solution of 1 (27 mg, 24 μmol) in DCM (6 mL) isdegassed by bubbling N₂ through the solution. While N2 is still bubbledthrough the solution, the degassed solution of pyrrolidine is added. Theweighed Pd(PPh₃)₄ is dissolved in DCM (1 mL) and 0.9 mL of this solutionis added. After 50 min of bubbling of N2, DCM (25 mL) is added and themixture is washed with aqueous saturated NH₄Cl (25 mL). Afterseparation, the aqueous layer is extracted with DCM (2×25 mL). Thecombined organic layers are dried (Na₂SO₄) and concentrated. The residueis purified by RP-HPLC (30-90% MeCN (0.1% formic acid) in H₂O (0.1%formic acid). The combined fractions are passed through SPE (HCO₃)columns and concentrated. After addition of MeCN (50 mL) the mixture isagain concentrated. The resulting residue 2 is used in the next step.

The conversion of the reaction can be monitored through LCMS analysis.Column: XBridge BEH C18 Intelligent Speed (IS) Column, 130 Å, 3.5 μm(4.6 mm×20 mm). Mobile phase A: Water (0.1% formic acid), Mobile phase B(0.1% formic acid). Detection with PDA and ESI+. Samples can be preparedby diluting the reaction mixture with MeCN.

(b) To a solution of the above residue 2 in CHCl₃ (5 mL) is added asolution of 3 (15 mg, 36 μmol, mw 418 g/mole) in CHCl₃ (0.8 mL). Theresulting mixture is added to solid EDC.HCl (4.7 mg, 25 μmol), CHCl₃ (5mL) was added and the mixture stirred for 30 minutes. DCM (30 mL) isadded and the resulting mixture is washed with water (30 mL). Afterseparation, the aqueous phase is extracted with DCM (30 mL). Thecombined organic layers are dried (Na₂SO₄) and concentrated. The residueis purified by RP-HPLC (30-90% MeCN (no acid) in H₂O (0.01% formicacid). The HPLC collection tubes are filled with 5% aqueous (NH₄)HCO₃before collection. The combined HPLC fractions are extracted with DCM(3×20 mL). The combined organic layers are dried (Na₂SO₄) andconcentrated. The product 4 is obtained as slightly yellow/white oil (21mg, 16 μmol, mw 1323 g/mole, 67% over two steps).

The conversion of the reaction can be monitored through LCMS analysis.Column: XBridge BEH C18 Intelligent Speed (IS) Column, 130 Å, 3.5 μm(4.6 mm×20 mm). Mobile phase A: Water (0.1% formic acid), Mobile phase B(0.1% formic acid). Detection with PDA and ESI+.

Glycan Remodelling (Anti-DLK1 ADC, ConjA1)

Antibody Preparation

Approximately 60 mg of anti-DLK1 antibody was buffer exchanged into 25mM Tris/Cl, 150 mM NaCl, pH 8.0 via a G25 desalting column; 4×2.5 mL at6 mg/mL loaded onto 4×PD10 desalting columns (GE 17085101). The bufferexchanged antibody was then concentrated to at least 25 mg/mL using aVivaspin 20 centrifugal concentrator (Sigma Z614637). The proteinconcentration was confirmed as 28.4 mg/mL by A280-320 nm UV analysisusing an extinction coefficient of 1.5.

Remodelling Reaction

Glycan remodelling was performed in a single pot reaction overnight (16hours) at room temperature. The following reaction mixture was preparedwith the solutions/reagents added in the order detailed below:

Solution Volume (uL) Concentration Final 25 mM Tris/Cl, 150 mM NaCl,663.8 NA pH 8.0 anti-DLK1 antibody in 25 mM 2000 15 mg/mL Tris/Cl, 150mM NaCl, pH 8.0 3.5 mg/mL EndoSH in 25 mM 165.7 1% w/w relative Tris/Cl,pH 8.0 to mAb 4.82 mg/mL His-TnGalNAcT in 902.5 7.5% w/w relative 25 mMTris/Cl, pH 8.0 to mAb 1M MgCl₂ in MQ water 38.7 10 mM 0.1M6-N₃GalNAc-UDP 96 25 Eq relative in MQ water to mAb

Protein A Purification Procedure

Protein A bind and elute was performed on a 5 mL HiTrap MabSelect Surecolumn (GE 11-0034-94). All chromatography steps were performed at 240cm/hr flow rate using an AKTA Prime plus system. The column was preparedand used as follows:

Solution CVs Notes 0.1M NaOH  3 Sanitization 25 mM Tris/Cl, 25 mM NaCl,10 Equilibration pH 8.0 anti-DLK1 antibody Sample As required Load 0.4MPotassium Phosphate, 20 Wash/Endotoxin 0.2% Triton 100, pH 7.0 removalstep 25 mM Tris/Cl, 25 mM NaCl, 20 Wash/Triton pH 8.0 removal step 0.15MAcetic Acid As required Collect peak 0.1 to 0.1 AU

After protein A affinity purification, a small sample of the product maybe reduced with DTT and subsequently subjected to MS analysis. A typicalmass spectrum of a successful transfer reaction shows the formation of aone major product of (90% of total heavy chain), resulting from modifiedgalactose transfer to core GlcNAc(Fuc) substituted Ab, and a minorproduct (±10% of total heavy chain), resulting from modified galactosetransfer to core GlcNAc (without Fucose) substituted antibody.

Post Protein a Buffer Exchange

The remodeled & purified anti-DLK1-GalNAc-N3 antibody was then bufferexchanged into phosphate buffered saline (PBS) and concentrated toapproximately 16.6 mg/mL using a Vivaspin 20 centrifugal concentrator(Sigma Z614637). The protein A eluate was diluted 1:1 with PBS and thenconcentrated back to the original volume and this repeated 6 times. Thevolume was finally reduced to target 16-17 mg/mL and the samplerecovered from the device. The protein concentration was confirmed as16.4 mg/mL by A280-320 nm UV analysis using an extinction coefficient of1.5 and a total of 2.7 mL was recovered.

Conjugation of 4 to Modified Antibody to Produce ConjA1

Reaction Conditions

-   -   2.7 mL of 16.4 mg/mL azido-modified DLK1 antibody (in this        example, HuBa-1-3d as described herein)    -   0.3 mL of 10 mM 4 in Dimethylacetamide

The reaction was mixed thoroughly and left to conjugate overnight (16hours) at room temperature. The conjugation mixture was filtered througha 0.2 μm PVDF filer (Millipore SLGV033RS) prior to final purificationand formulation.

Purification of ConjA1

The filtered conjugation mixture was purified using a Vivaspin 20centrifugal concentrator (Sigma Z614637). The conjugation mixture wasdiluted 1:1 with 30 mM Histidine HCl, 200 mM Sorbitol, pH 6.0 and theconcentrated back to the original volume. This was repeated 12 timesbefore the purified ADC bulk was recovered from the centrifugal device.

The protein concentration was determined by quantitative SEC analysisusing a calibration curve of the antibody and the conjugate diluted toapproximately 5 mg/mL with additional 30 mM Histidine HCl, 200 mMSorbitol, pH 6.0. Tween 20 was added to 0.02% w/v from a 1% stock in 30mM Histidine HCl, 200 mM Sorbitol, pH 6.0 and the concentration retestedby quantitative SEC analysis. A sample was taken for testing and theremainder split into 1 mL aliquots and frozen at −80 C.

Analysis of the product showed the following properties:

-   -   Clear, colourless, particulate free    -   5.17 [protein] by SEC    -   Average DAR of 1.87 (by RP chromatography)    -   0.09 EU/mg endotoxin    -   ≤4.5% free drug-linker    -   pH 6.06

Glycan Remodelling (Anti-KAAG1 ADC, ConjA2)

Antibody Preparation

Approximately 150 mgs of anti-KAAG1 (approximately 25 mL at 6.13 mg/mLin PBS pH 7.4) was buffer exchanged into 25 mM Tris/Cl, 150 mM NaCl, pH8.0 and concentrated to >25 mg/mL using Vivaspin 20 centrifugalconcentrators (Sigma Z614637). Initially, the antibody was concentratedto 12 mL and then diluted 1:1 with 25 mM Tris/Cl, 150 mM NaCl, pH 8.0and then concentrated back to 12 mL and this process repeated 6 times.Finally, the buffer exchanged stock was further concentrated to 6 mL.The protein concentration was determined by A280-320 nm UV analysisusing an extinction coefficient of 1.5 and then diluted to 25 mg/mL with25 mM Tris/Cl, 150 mM NaCl, pH 8.0.

Remodelling Reaction

Glycan remodelling was performed in a single pot reaction overnight (16hours) at room temperature. The following reaction mixture was preparedwith the solutions/reagents added in the order detailed below:

Solution Volume (uL) Concentration Final 25 mM Tris/Cl, 150 mM NaCl, 893NA pH 8.0 Anti-KAAG1 mAb in 25 mM 6000 15 mg/mL Tris/Cl, 150 mM NaCl, pH8.0 3.5 mg/mL EndoSH in 25 428.4 1% w/w relative mM Tris/Cl, pH 8.0 tomAb 4.82 mg/mL His-TnGalNAcT 2330 7.5% w/w relative in 25 mM Tris/Cl, pH8.0 to mAb 1M MgCl2 in MQ water 100.2 10 mM 0.1M 6-N3GalNAc-UDP in 25025 Eq relative MQ water to mAb

Protein A Purification Procedure

Protein A bind and elute was performed on a 4.7 mL HiScreen MabSelectSure column (GE 28-9269-77). All chromatography steps were performed at240 cm/hr flow rate using an AKTA Prime plus system. The column wasprepared and used as follows:

Solution CVs Notes 0.1M NaOH  3 Sanitization 25 mM Tris/Cl, 25 mM NaCl,10 Equilibration pH 8.0 Antibody Sample As required Load 0.4M PotassiumPhosphate, 20 Wash/Endotoxin 0.2% Triton 100, pH 7.0 removal step 25 mMTris/Cl, 25 mM NaCl, 20 Wash/Triton pH 8.0 removal step 0.15M AceticAcid As required Collect peak 0.1 to 0.1 AU

After protein A affinity purification, a small sample of the product maybe reduced with DTT and subsequently subjected to MS analysis. A typicalmass spectrum of a successful transfer reaction shows the formation of aone major product of (90% of total heavy chain), resulting from modifiedgalactose transfer to core GlcNAc(Fuc) substituted Ab, and a minorproduct (±10% of total heavy chain), resulting from modified galactosetransfer to core GlcNAc (without Fucose) substituted antibody.

Post Protein A Buffer Exchange

The protein A eluate containing the remodeled/purified Ab-GalNAc-N3 waspH adjusted with the addition of 1.5M Tris base at 3.2% v/v and thenbuffer exchanged into PBS and concentrated to ≈17 mg/mL using Vivaspin20 centrifugal concentrators (Sigma Z614637). Initially, the pH adjustedpool was diluted 1:1 PBS and then concentrated back to the originalvolume and this process repeated 6 times. Finally, the buffer exchangedstock was further concentrated to target≈17 mg/mL. The proteinconcentration was confirmed at 16.5 mg/mL by A280-320 nm UV analysisusing an extinction coefficient of 1.5; a total of 7.9 mL was recoveredfor a yield of 88%.

Conjugation of 4 to Modified Antibody to Produce ConjA2

To 7.9 mL of 16.5 mg/mL Ab-GalNAc-N3 (Ab=3A4, as described herein) wasadded 0.788 mL of 10 mM PL1601 in DMF (10% final v/v DMF). The reactionwas mixed thoroughly and left to conjugate overnight (16 hours) at roomtemperature. The conjugation mixture was filtered through a 0.22 μm PESfilter (Millipore SLGV033RS) prior to final purification andformulation.

Purification of ConjA2

The filtered conjugation mixture was purified by constant-volumediafiltration using a 30 kDa Pellicon 3 membrane at ≈50 g/m² of membranearea, a crossflow of 5.0±0.25 L/min/m2, TMP of 1.0±0.2 bar and a totalof 12 diavolumes of buffer exchange into PBS pH7.4. The diafiltered poolwas recovered from the UFDF and filtered through a 0.22 μm PES membranefilter (Millipore SLGV033RS) into sterile eppendorfs. The proteinconcentration was determined by A280-320 nm UV analysis using anextinction coefficient of 1.5 and was determined to be 4.9 mg/mL. Asample was taken for testing and the remainder stored at 4° C.

Analysis of the product showed the following properties:

-   -   Clear, colourless, particulate free    -   4.9 mg/ml [protein] by A280/330 nm spectroscopy    -   Average DAR of 1.9 (by RP chromatography)    -   0.07 EU/mg endotoxin    -   3% free drug-linker    -   98.3% monomer by sixe exclusion chromatography

Glycan Remodelling (Anti-Mesothelin ADC, ConjA3)

Antibody Preparation

Approximately 60 mg of anti-Mesothelin antibody is buffer exchanged into25 mM Tris/Cl, 150 mM NaCl, pH 8.0 via a G25 desalting column; 4×2.5 mLat 6 mg/mL loaded onto 4×PD10 desalting columns (GE 17085101). Thebuffer exchanged antibody is then concentrated to at least 25 mg/mLusing a Vivaspin 20 centrifugal concentrator (Sigma Z614637). Theprotein concentration is confirmed by A280-320 nm UV analysis using anextinction coefficient of 1.5.

Remodelling Reaction

Glycan remodelling is performed in a single pot reaction overnight (16hours) at room temperature. The following reaction mixture is preparedwith the solutions/reagents added in the order detailed below:

Solution Volume (uL) Concentration Final 25 mM Tris/Cl, 150 mM NaCl,663.8 NA pH 8.0 anti-Mesothelin antibody in 2000 15 mg/mL 25 mM Tris/Cl,150 mM NaCl, pH 8.0 3.5 mg/mL EndoSH in 25 mM 165.7 1% w/w relativeTris/Cl, pH 8.0 to mAb 4.82 mg/mL His-TnGalNAcT 902.5 7.5% w/w relativein 25 mM Tris/Cl, pH 8.0 to mAb 1M MgCl₂ in MQ water 38.7 10 mM 0.1M6-N₃GalNAc-UDP in 96 25 Eq relative MQ water to mAb

Protein A Purification Procedure

Protein A bind and elute is performed on a 5 mL HiTrap MabSelect Surecolumn (GE 11-0034-94). All chromatography steps are performed at 240cm/hr flow rate using an AKTA Prime plus system. The column is preparedand used as follows:

Solution CVs Notes 0.1M NaOH  3 Sanitization 25 mM Tris/Cl, 25 mM NaCl,10 Equilibration pH 8.0 anti-Mesothelin antibody As required Load Sample0.4M Potassium Phosphate. 20 Wash/Endotoxin 0.2% Triton 100, pH 7.0removal step 25 mM Tris/Cl, 25 mM NaCl, 20 Wash/Triton pH 8.0 removalstep 0.15M Acetic Acid As required Collect peak 0.1 to 0.1 AU

After protein A affinity purification, a small sample of the product maybe reduced with DTT and subsequently subjected to MS analysis. A typicalmass spectrum of a successful transfer reaction shows the formation of aone major product of (90% of total heavy chain), resulting from modifiedgalactose transfer to core GlcNAc(Fuc) substituted Ab, and a minorproduct (±10% of total heavy chain), resulting from modified galactosetransfer to core GlcNAc (without Fucose) substituted antibody.

Post Protein a Buffer Exchange

The remodeled & purified anti-Mesothelin-GalNAc-N3 antibody is thenbuffer exchanged into phosphate buffered saline (PBS) and concentratedto approximately 16.6 mg/mL using a Vivaspin 20 centrifugal concentrator(Sigma Z614637). The protein A eluate is diluted 1:1 with PBS and thenconcentrated back to the original volume and this is repeated 6 times.The volume is finally reduced to target 16-17 mg/mL and the samplerecovered from the device. The protein concentration is confirmed byA280-320 nm UV analysis using an extinction coefficient of 1.5.

Conjugation of 4 to Modified Antibody to Produce ConjA3

Reaction Conditions

-   -   2.7 mL of ˜15 mg/mL azido-modified anti-Mesothelin antibody    -   0.3 mL of 10 mM 4 in Dimethylacetamide

The reaction is mixed thoroughly and left to conjugate overnight (16hours) at room temperature. The conjugation mixture is filtered througha 0.2 μm PVDF filer (Millipore SLGV033RS) prior to final purificationand formulation.

Purification of ConjA3

The filtered conjugation mixture is purified using a Vivaspin 20centrifugal concentrator (Sigma Z614637). The conjugation mixture isdiluted 1:1 with 30 mM Histidine HCl, 200 mM Sorbitol, pH 6.0 and thenconcentrated back to the original volume. This is repeated 12 timesbefore the purified ADC bulk is recovered from the centrifugal device.

The protein concentration is determined by quantitative SEC analysisusing a calibration curve of the antibody and the conjugate diluted toapproximately 5 mg/mL with additional 30 mM Histidine HCl, 200 mMSorbitol, pH 6.0. Tween 20 is added to 0.02% w/v from a 1% stock in 30mM Histidine HCl, 200 mM Sorbitol, pH 6.0 and the concentration retestedby quantitative SEC analysis. A sample is taken for testing and theremainder split into 1 mL aliquots and frozen at −80 C.

In Vitro Cytotoxicity of ConjA1

Flasks of either Lu135 cell or SK-N-FI cells were trypsin treated andthe liberated cells were washed and re-suspended in fresh medium. Thecell density was determined by mixing 1:1 with Trypan blue (0.4% (w/v)Sigma TB154) and counting clear/blue (live/dead) cells with a Luna IIautomated cell counter (Logos Biosystems). The cell suspension wasdiluted to the required seeding density (20×104/m1), dispensed intowhite 96-well flat bottomed microplates (50 μl/well) and incubatedovernight.

A stock solution (1 ml) of ConjA1 (20 μg/ml) was made by dilution offilter-sterile ConjA1 into the same cell culture medium. A set of 8×10-fold dilutions of stock ConjA1 was made in a sterile 24-well plate byserial transfer of 100 μl into 900 μl of cell culture medium. EachConjA1 dilution was dispensed, 50 μl/well, into 4 replicate wells of the96-well plate, containing cells suspension. Control wells received thesame volume of culture medium only.

After the ConjA1 exposure period, cell viability was measured by PromegaCellTiter-Glo by adding 100 μl/well, agitate for 2 mins and read on theEnvision using the Luminescence protocol. Data were analysed usingGraphpad Prism software.

The EC₅₀ of ConjA1 against Lu-135 cells was found to be 0.01765 μg/mL.The EC₅₀ of the ADC control was 0.5326 μg/mL (see FIG. 1A*).

The EC₅₀ of ConjA1 against SK-N-FI cells was found to be 0.1565 μg/mL.The EC₅₀ of the ADC control was 5×10⁵ μg/mL (see FIG. 1B*). *ln bothFIGS. 1A*1B, ▾ is ConjA1 and ● is an otherwise identical ADC controlcomprising the non-DLK1 specific antibody B12).

In Vivo Efficacy Studies with ConjA1

In vivo anti-tumor activity in liver cancer patient-derived xenograft(PDX) LI1097 model LI1097 seed tumours were revived subcutaneously inNOD/SCID mice, and maintained subcutaneously in BALB/c nude mice beforeimplantation. When the tumour volumes reached 700-1500 mm³, tumours werecollected and cut into pieces of about 2-3 mm3 in diameter. The tumoursor tumour pieces were washed with ice cold RPM11640 media (withoutserum) and subsequently placed into ice cold media for use.

The skin of five-to-six week old, female BALB/c nude mice wasdisinfected at the right flank by iodophor before tumour implantation.Each mouse was inoculated without anesthesia, subcutaneously at theright upper flank with one primary human liver cancer LI1097 tumourfragment for tumour development.

After tumour inoculation, the animals were checked daily for morbidityand mortality. Tumour size was measured by caliper twice weekly in twodimensions. The tumour volume was expressed in mm³ using the formula:TV=0.5 a×b2 where a and b are the long and short diameters of thetumour, respectively.

On study day 12, mice were randomised into 5 groups of 8 mice each; themean tumour volume was ˜170 mm³ across the cohort. Mice were dosed withthe test agents on study day 13 (day 1, indicated by vertical dottedline in the graph). Test mice in this study received a single dose oftheir allocated test article and dose level on day 1 and tumour growthwas monitored thereafter, up to day 51.

The results are shown in FIG. 2A, where:

-   -   ∘=vehicle, qd×1    -   ⋄=ADC control using non-DLK1 specific antibody B12, 1 mg/kg,        qd×1    -   □=ConjA1, 0.1 mg/kg, qd×1    -   Δ=ConjA1, 0.3 mg/kg, qd×1    -   ▾=ConjA1, 1.0 mg/kg, qd×1

As is clear from FIG. 2A, ConjA1 at 1.0 mg/kg led to the greatestslowing of tumour growth, followed by the ConjA1 at 0.3 mg/kg. Moreover,at the highest dose tested, ConjA1 resulted in 3/8 PR and 2/8 CR, whilenone of the mice treated with the vehicle or the isotype-control ADC (1mg/kg, single dose) had any PR, CR or TFS.

In Vivo Anti-Tumor Activity in the Neuroblastoma SK-N-FI Xenograft Model

Female, NOD-SCID mice were six weeks old on day of implant. SK-N-FIcells were harvested during log phase growth and resuspended inphosphate buffered saline with 50% matrigel. Using a 26 G syringe, 100μL (3×10⁶ cells) of the cell suspension mixture was subcutaneouslyinjected into the right flank of each mouse. Animals were examined twiceper week with their body weight and tumor size. Tumor size was measuredusing digital calipers and calculated according to the followingexpression:Tumor volume (mm³)=(minor axis) 2×(major axis)×π/6

Eighteen days after transplant of the cancer cells, 50 mice whose tumorvolume was between 99.0 mm³ and 155.2 mm³ (average 116.2 mm³) weredivided into 5 groups (N=10 in each group). On the dosing day, testsubjects were administrated by intravenous injection from tail vein. Theend point of the study was set as when each tumor reaches the endpointvolume of 1000 mm³ or at the end of the study (60 days after dosing),whichever came first.

The results are shown in FIG. 2B, where:

⋄=vehicle, qd×1

∘=ADC control using non-DLK1 specific antibody B12, 0.5 mg/kg, qd×1

□=ADC control using non-DLK1 specific antibody B12, 1 mg/kg, qd×1

Δ=ConjA1, 0.5 mg/kg, qd×1

▾=ConjA1, 1.0 mg/kg, qd×1

As is clear from FIG. 2B, ConjA1 at 1.0 mg/kg led to the greatestslowing of tumour growth, followed by the ConjA1 at 0.5 mg/kg.

In the human neuroblastoma-derived SK-N-FI xenograft model a single doseof ConjA1 at 0.5 or 1 mg/kg showed dose-dependent anti-tumor activitycompared to the vehicle- and isotype control ADC-treated mice.

At the highest dose tested, ConjA1 resulted in 1/9 partial responders(PR) and 4/9 complete responder (CR), one of which was a tumor-freessurvivor (TFS) at the end of the study on day 60 (one animal of theinitial 10 in this group was excluded fro excluded from the finalfigures for treatment-unrelated reasons).

Rat Toxicology Study

Rat toxicology study (off-target toxicity assessment, tested ADC doesnot bind rat Dlk-1)

Method ConjA1 was evaluated in a single intravenous dose rattolerability study. Male sprague-dawley rats (n=3/group) were dosed at 5mg/kg on day 1, with necropsy on day 21 following dosing. Bodyweightsand food consumption were monitored frequently with in-life sampling forclinical pathology (blood on days 8 and 21) and repeated sampling forpharmacokinetics. At necropsy, macroscopic observations were taken withselected organs weighed and retained for possible histopathology.

Results

ConjA1 was clinically well tolerated at 5 mg/kg with no marked adverseclinical signs. Bodyweight gain was reduced, with the animals beingaround 15% lighter than the control group at the end of the study. Whiteblood cell count was reduced on day 8 (neutrophils reduced by around 95%compared to concurrent control), with evidence of recovery by day 22.

Overall Conclusion

ConjA1 was well stable, well tolerated and showed a favorablepharmacokinetic profile in the rat with a half-life of 9 days at 5mg/kg. This suggest that the MTD in rats is at least 5 mg/kg or higher.

In Vitro Cytotoxicity in A204 and Hep3B Cells in 2D and 3D Cell Culture

Materials & Methods

Cells were seeded in onco-media (RPMI, 5% FBS, 2 mML-alanyl-L-glutamine, 1 mM sodium pyruvate and 1%penicillin/streptomycin) in 384-well Elplasia plates pre-coated withpHEMA.

The ADC, ConjA1, and B12-1601 compounds were added 24 hours post cellseeding, with a starting concentration of 10 mg/mL, a 10-fold serialdilution across 9 concentrations in quadruplicate. The incubation timeswith the ADC's compounds were 5 days in 2D- and 7 days in 3D-cultureswith media exchanges every 3 days, for a total of 14.

At the end of incubation period, cells were lysed and analyzed todetermine cell viability. Cell proliferation end-point was analyzed asPercent of Control (POC) using the following formula:POC=relative cell count (compound wells)/relative cell count (vehiclecontrol wells)×100%

Data were analysed using Graphpad Prism software.

Results

FIG. 3 shows data from in vitro cytotoxicity in A204 and Hep3B cells in3D cell culture:

-   -   FIG. 3A is the A204 cell data [IC₅₀ 3 D ADC=0.001460 μg/ml, IC₅₀        3 D B12-PL1601=0.7662 μg/ml, IC₅₀ 2D ADC=0.006399 μg/ml, IC₅₀ 3D        B12-PL1601=0.4059 μg/m];    -   FIG. 3B is the Hep3B cell data [IC₅₀ 3 D ADC=0.2271 μg/ml, IC₅₀        3 D B12-PL1601=˜432.6 μg/ml, IC₅₀ 2D ADC=˜59.29 μg/ml, IC₅₀ 2D        B12-PL1601=˜3.957 μg/ml].

In Vitro Cytotoxicity of ConjA2

Flasks of either SN12C and MDA-MB-231 FI cells were trypsin treated andthe liberated cells were washed and re-suspended in fresh medium. Thecell density was determined by mixing 1:1 with Trypan blue (0.4% (w/v)Sigma TB154) and counting clear/blue (live/dead) cells with a Luna IIautomated cell counter (Logos Biosystems). The cell suspension wasdiluted to the required seeding density (20×10⁴/ml), dispensed intowhite 96-well flat-bottomed microplates (50 μl/well), and incubatedovernight.

A stock solution (1 ml) of ConjA2 (20 μg/ml) was made by dilution offilter-sterile ConjA2 into the same cell culture medium. A set of 8×10-fold dilutions of stock ConjA2 was made in a sterile 24-well plate byserial transfer of 100 μl into 900 μl of cell culture medium. EachConjA2 dilution was dispensed, 50 μl/well, into 4 replicate wells of the96-well plate, containing cells suspension. Control wells received thesame volume of culture medium only.

After the ConjA2 exposure period, cell viability was measured by PromegaCellTiter-Glo by adding 100 μl/well, agitate for 2 mins and read on theEnvision using the Luminescence protocol. Data were analysed usingGraphpad Prism software.

The EC₅₀ of ConjA2 against SN12C cells was found to be 0.0663 μg/mL. TheEC₅₀ of the ADC control was not detectable (see FIG. 4A*).

The EC₅₀ of ConjA2 against MDA-MB-231 cells was found to be 0.226 μg/mL.The EC₅₀ of the ADC control was again not detectable (see FIG. 4B*). Inboth FIGS. 4A & 4B, ♦ is ConjA2 and ● is an otherwise identical ADCcontrol comprising the non-KAAG1 specific antibody B12).

In Vivo Efficacy Study of ConjA2

Female athymic nude mice (Crl:NU(Ncr)-Foxn1nu, Charles River) were eightweeks old with a body weight (BW) range of 20.7-31.2 g on Day 1 of thestudy.

On the day of implant, MDA-MB-231 tumor cells used for implantation wereharvested during log phase growth and resuspended in phosphate-bufferedsaline (PBS) at 5×10⁷ cells/mL. Each mouse was injected subcutaneously(s.c.) in the right flank with 5×10⁶ cells (0.1 mL cell suspension) andtumors were monitored as their volumes approached the target range of100 to 150 mm³. Tumors were measured in two dimensions using calipers,and volume was calculated using the formula:Tumor Volume (mm³)=w²×l/2where w=width and l=length, in mm, of the tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Sixteen days after tumor implantation, designated as Day 1 of the study,the animals were sorted into groups each consisting of 8 mice withindividual tumor volumes of 108 to 144 mm³ and group mean tumor volumesof 112.5-123.8 mm³. On Day 1 of the study, all treatments wereadministered intravenously (i.v.) in a single injection (qd×1) via tailvein injection in a dosing volume of 0.2 mL per 20 grams of body weight(10 mL/kg), scaled to the body weight of each individual animal. Tumorswere measured using calipers twice per week, and each animal waseuthanized when its tumor reached the endpoint volume of 1500 mm³ or atthe end of the study, whichever came first. The study ended on Day 59.

The results are shown in FIG. 5, where:

-   -   ∘=vehicle, qd×1 (upper line)    -   □=ADC control using non-KAAG1 specific antibody B12, 0.6 mg/kg,        qd×1    -   ∘=ConjA2, 0.6 mg/kg, qd×1 (lower line)

As is clear from FIG. 5, ConjA2 at 0.6 mg/kg led to significant slowingof tumour growth.

In Vivo Anti-Tumor Activity of ConjA2 in SN12C Xenograft Model

Female severe combined immunodeficient mice (Fox Chase SCID®,CB17/lcr-Prkdcscid/IcrlcoCrl, Charles River) were nine weeks old with abody weight (BW) range of 15.4 to 22.2 g on Day 1 of the study.

On the day of tumor implant, each test mouse received 5×106 SN12C cells(0.1 mL cell suspension in 50% Matrigel® Matrix (Corning®) in phosphatebuffered saline) implanted subcutaneously in the right flank. Tumorgrowth was monitored as the average size approached the target range of100 to 150 mm³. Tumors were measured in two dimensions using calipers,and volume was calculated using the formula:Tumor Volume (mm³)=w2×l/2

where w=width and l=length, in mm, of the tumor. Tumor weight may beestimated with the assumption that 1 mg is equivalent to 1 mm³ of tumorvolume.

Twenty-three days after tumor implantation, designated as Day 1 of thestudy, the animals were sorted into nine groups (n=8) with individualtumor volumes of 108 to 172 mm³ and group mean tumor volumes of 129 mm³.

On Day 1 of the study, all treatments were administered intravenously(i.v.) in a single injection (qd×1) via tail vein injection in a dosingvolume of 0.2 mL per 20 grams of body weight (10 mL/kg), scaled to thebody weight of each individual animal. Tumors were measured usingcalipers twice per week, and each animal was euthanized when its tumorreached the endpoint volume of 1000 mm³ or at the end of the study,whichever came first. The study ended on Day 60.

The data is shown in FIG. 6, where it can be seen that administration ofthe ADC (ConjA2) reduced tumour growth in a dose-dependent manner.

In Vitro Cytotoxicity

Flasks of either OVCAR3, CAPAN-2 or HPAC cells are trypsin treated andthe liberated cells are washed and re-suspended in fresh medium. Thecell density is determined by mixing 1:1 with Trypan blue (0.4% (w/v)Sigma TB154) and counting clear/blue (live/dead) cells with a Luna IIautomated cell counter (Logos Biosystems). The cell suspension isdiluted to the required seeding density (20×104/m1), dispensed intowhite 96-well flat bottomed microplates (50 μl/well) and incubatedovernight.

A stock solution (1 ml) of ConjA3 (20 μg/ml) is made by dilution offilter-sterile ConjA3 into the same cell culture medium. A set of 8×10-fold dilutions of stock ConjA3 is made in a sterile 24-well plate byserial transfer of 100 μl into 900 μl of cell culture medium. EachConjA3 dilution is dispensed, 50 μl/well, into 4 replicate wells of the96-well plate, containing cells suspension. Control wells receive thesame volume of culture medium only.

After the ConjA3 exposure period, cell viability is measured by PromegaCellTiter-Glo by adding 100 μl/well, agitate for 2 mins and read on theEnvision using the Luminescence protocol. Data are analysed usingGraphpad Prism software.

In Vivo Efficacy Study of ConjA3

In Vivo Anti-Tumor Activity in a OVCAR3 Model

Seed tumours are revived subcutaneously in NOD/SCID mice, and maintainedsubcutaneously in BALB/c nude mice before implantation. When the tumourvolumes reached 700-1500 mm³, tumours are collected and cut into piecesof about 2-3 mm3 in diameter. The tumours or tumour pieces are washedwith ice cold RPM11640 media (without serum) and subsequently placedinto ice cold media for use.

The skin of five-to-six week old, female BALB/c nude mice is disinfectedat the right flank by iodophor before tumour implantation. Each mouse isinoculated without anesthesia, subcutaneously at the right upper flankwith one tumour fragment for tumour development.

After tumour inoculation, the animals are checked daily for morbidityand mortality. Tumour size is measured by caliper twice weekly in twodimensions. The tumour volume is expressed in mm³ using the formula:TV=0.5 a×b2 where a and b are the long and short diameters of thetumour, respectively.

On study day 12, mice are randomised into 5 groups of 8 mice each; thetarget mean tumour starting volume is ˜170 mm³ across the cohort. Miceare dosed with the test agents on study day 13. Test mice in this studyreceive a single dose of their allocated test article and dose level onday 1 and tumour growth is monitored thereafter, up to day 51.

SEQUENCES

SEQUENCES SEQ ID NO. 1 [HuBa-1-3d VH, CDR underline]QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVT VSSSEQ ID NO. 2 [HuBa-1-3d VL, CDR underline]DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPPTFGQGTKLEIKSEQ ID NO. 3 [HuBa-1-3d Heavy Chain]QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 4 [HuBa-1-3d Light Chain]DIVMTQSPDSLAVSLGERATINCKSSQSLLNSSNQKNYLAWYQQKPGQPPKLLVYFASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYSTPPTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 5 [HuBa-1-3d VH CDR1]DYAMH SEQ ID NO. 6 [HuBa-1-3d VH CDR2] VISTYYGNTNYNQKFKGSEQ ID NO. 7 [HuBa-1-3d VH CDR3] GGLREYYYAMDYSEQ ID NO. 8 [HuBa-1-3d VL CDR1] KSSQSLLNSSNQKNYLASEQ ID NO. 9 [HuBa-1-3d VL CDR2] FASTRESSEQ ID NO. 10 [HuBa-1-3d VL CDR3] QQHYSTPPTSEQ ID NO. 11 [HuBa-1-3d Heavy Chain, terminal K]QVQLVQSGAEVKKPGASVKVSCKGSGYTFTDYAMHWVRQAPGQGLEWIGVISTYYGNTNYNQKFKGKATMTVDKSTSTAYMELRSLRSDDTAVYYCARGGLREYYYAMDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297SEQ ID NO. 12 [Human DLK1, variant 1]MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCHVGWQGPLCDQCVTSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNGTCVSLDGGLYECSCAPGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCTPNPCENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQVSYECLCKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELNKKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQYNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI SEQ ID NO. 13 [Human DLK1, variant 2]MTATEALLRVLLLLLAFGHSTYGAECFPACNPQNGFCEDDNVCRCQPGWQGPLCDQCVTSPGCLHGLCGEPGQCICTDGWDGELCDRDVRACSSAPCANNRTCVSLDDGLYECSCAPGYSGKDCQKKDGPCVINGSPCQHGGTCVDDEGRASHASCLCPPGFSGNFCEIVANSCTPNPCENDGVCTDIGGDFRCRCPAGFIDKTCSRPVTNCASSPCQNGGTCLQHTQVSYECLCKPEFTGLTCVKKRALSPQQVTRLPSGYGLAYRLTPGVHELPVQQPEHRILKVSMKELNKKTPLLTEGQAICFTILGVLTSLVVLGTVGIVFLNKCETWVSNLRYNHMLRKKKNLLLQYNSGEDLAVNIIFPEKIDMTTFSKEAGDEEI SEQ ID NO. 101 [3A4 VH, CDR underline]QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSSEQ ID NO. 102 [3A4 VL, CDR underline]DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKSEQ ID NO. 103 [3A4 Heavy Chain]QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 104 [3A4 Light Chain]DIVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPQLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 105 [3A4 VH CDR1]GYTFTDDYMS SEQ ID NO. 106 [3A4 VH CDR2] DIN PYNGDTNSEQ ID NO. 107 [3A4 VH CDR3] DPGAMDY SEQ ID NO. 108 [3A4 VL CDR1]RSSQSLLHSNGNTYLE SEQ ID NO. 109 [3A4 VL CDR2] TVSNRFSSEQ ID NO. 110 [3A4 VL CDR3] FQGSHVPLTSEQ ID NO. 111 [3A4 Heavy Chain, terminal K]QIQLVQSGAEVKKPGASVKVSCKASGYTFTDDYMSWVKQAPGQGLEWIGDINPYNGDTNYNQKFKGKATLTVDKSTSTAYMELSSLRSEDTAVYYCARDPGAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297 SEQ ID NO. 112 [Human KAAG1]MDDDAAPRVEGVPVAVHKHALHDGLRQVAGPGAAAAHLPRWPPPQLAASRREAPPLSQRPHRTQGAGSPPETNEKLTNPQVKEK SEQ ID NO. 113 [3A4-L2 VL, CDR underline]DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKWYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKSEQ ID NO. 114 [3A4-L2 Light Chain]DVVMTQTPLSLPVTPGEPASISCRSSQSLLHSNGNTYLEWYLQKPGQSPKLLIYTVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPLTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO. 115 [3A4-K4 VL, CDR underline]DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKWYTVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIKSEQ ID NO. 116 [3A4-K4 Light Chain]DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLEWYQQKPGQPPKWYTVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGSHVPLTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO. 201 [XA4 VH, CDR underline]QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVT VSSSEQ ID NO. 202 [XA4 VL, CDR underline]EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKSEQ ID NO. 203 [XA4 Heavy Chain]QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 204 [XA4 Light Chain]EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 205 [XA4 VH CDR1] IYGMHSEQ ID NO. 206 [XA4 VH CDR2] VLWYDGSHEYYADSVKGSEQ ID NO. 207 [XA4 VH CDR3] DGDYYDSGSPLDY SEQ ID NO. 208 [XA4 VL CDR1]RASQSVSSYLA SEQ ID NO. 209 [XA4 VL CDR21] DASNRATSEQ ID NO. 210 [XA4 VL CDR31] QQRSNWPLTSEQ ID NO. 211 [XA4 Heavy Chain, terminal K]QVHLVESGGGVVQPGRSLRLSCVASGITFRIYGMHWVRQAPGKGLEWVAVLWYDGSHEYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARDGDYYDSGSPLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297SEQ ID NO. 212 [XFT VH, CDR underline]QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSSEQ ID NO. 213 [XFT VL, CDR underline]DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIKSEQ ID NO. 214 [XFT Heavy Chain]QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 215 [XFT Light Chain]DIALTQPASVSGSPGQSITISCTGTSSDIGGYNSVSWYQQHPGKAPKLMIYGVNNRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSYDIESATPVFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 216 [XFT VH CDR1]GYSFTSYWIG SEQ ID NO. 217 [KFT VH CDR2] WMGIIDPGDSRTRYSPSFQGSEQ ID NO. 218 [XFT VH CDR3] GQLYGGTYMDG SEQ ID NO. 219 [XFT VL CDR1]TGTSSDIGGYNSVS SEQ ID NO. 220 [XFT VL CDR2] LMIYGVNNRPSSEQ ID NO. 221 [XFT VL CDR3] SSYDI ESATPSEQ ID NO. 222 [XFT Heavy Chain, terminal K]QVELVQSGAVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGKGLEWMGIIDPGDSRTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAMYYCARGQLYGGTYMDGWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297SEQ ID NO. 223 [X09 VH, CDR underline]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSSEQ ID NO. 224 [X09 VL, CDR underline]DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKSEQ ID NO. 225 [X09 Heavy Chain]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 226 [X09 Light Chain]DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 227 [X09 VH CDR1] GYSFTSEQ ID NO. 228 [X09 VH CDR2] LITPYNGASSYNQKFRGSEQ ID NO. 229 [X09 VH CDR3] GGYDGRGFDY SEQ ID NO. 230 [X09 VL CDR1]SASSSVSYMH SEQ ID NO. 231 [X09 VL CDR2] DTSKLASSEQ ID NO. 232 [X09 VL CDR3] QQWSKHPLTSEQ ID NO. 233 [X09 Heavy Chain, terminal K]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297SEQ ID NO. 234 [X09.2 VH, CDR underline]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGQGTTVTVSSSEQ ID NO. 235 [X09.2 VL, CDR underline]DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSGYPLTFGAGTKLEIKSEQ ID NO. 236 [X09.2 Heavy Chain]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG N* indicates Asn297SEQ ID NO. 237 [X09.2 Light Chain]DIELTQSPAIMSASPGEKVTMTCSASSSVSYMHWYQQKSGTSPKRWIYDTSKLASGVPGRFSGSGSGNSYSLTISSVEAEDDATYYCQQWSKHPLTFGSGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NO. 238 [X09.2 VH CDR1] GYTMNSEQ ID NO. 239 [X09.2 VH CDR2] LITPYNGASSYNQKFRGSEQ ID NO. 240 [X09.2 VH CDR3] GGYDGRGFDY SEQ ID NO. 241 [X09.2 VL CDR1]SASSSVSYMH SEQ ID NO. 242 [X09.2 VL CDR2] DTSKLASSEQ ID NO. 243 [X09.2 VL CDR3] QQWSGYPLTSEQ ID NO. 244 [X09.2 Heavy Chain, terminal K]QVQLQQSGPELEKPGASVKISCKASGYSFTGYTMNWVKQSHGKSLEWIGLITPYNGASSYNQKFRGKATLTVDKSSSTAYMDLLSLTSEDSAVYFCARGGYDGRGFDYWGSGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN*STYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK N* indicates Asn297SEQ ID NO. 245 [Human Mesothelin, variant 1]MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQAPRRPLPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLASEQ ID NO. 246 [Human Mesothelin, variant 2]MALPTARPLLGSCGTPALGSLLFLLFSLGWVQPSRTLAGETGQEAAPLDGVLANPPNISSLSPRQLLGFPCAEVSGLSTERVRELAVALAQKNVKLSTEQLRCLAHRLSEPPEDLDALPLDLLLFLNPDAFSGPQACTRFFSRITKANVDLLPRGAPERQRLLPAALACWGVRGSLLSEADVRALGGLACDLPGRFVAESAEVLLPRLVSCPGPLDQDQQEAARAALQGGGPPYGPPSTWSVSTMDALRGLLPVLGQPIIRSIPQGIVAAWRQRSSRDPSWRQPERTILRPRFRREVEKTACPSGKKAREIDESLIFYKKWELEACVDAALLATQMDRVNAIPFTYEQLDVLKHKLDELYPQGYPESVIQHLGYLFLKMSPEDIRKWNVTSLETLKALLEVNKGHEMSPQVATLIDRFVKGRGQLDKDTLDTLTAFYPGYLCSLSPEELSSVPPSSIWAVRPQDLDTCDPRQLDVLYPKARLAFQNMNGSEYFVKIQSFLGGAPTEDLKALSQQNVSMDLATFMKLRTDAVLPLTVAEVQKLLGPHVEGLKAEERHRPVRDWILRQRQDDLDTLGLGLQGGIPNGYLVLDLSMQEALSGTPCLLGPGPVLTVLALLLASTLA

The invention claimed is:
 1. A conjugate of formula (I):Ab-(DL)_(p)  (I) wherein: Ab is an antibody that binds to DLK1 andcomprises a VH domain comprising a VH CDR1 with the amino acid sequenceof SEQ ID NO.5, a VH CDR2 with the amino acid sequence of SEQ ID NO.6,and a VH CDR3 with the amino acid sequence of SEQ ID NO.7; and, a VLdomain comprising a VL CDR1 with the amino acid sequence of SEQ ID NO.8,a VL CDR2 with the amino acid sequence of SEQ ID NO.9, and a VL CDR3with the amino acid sequence of SEQ ID NO.10; wherein DL is

wherein: X is selected from the group comprising: a single bond, —CH2—and —C₂H₄—; n is from 1 to 8; m is 0 or 1; R⁷ is either methyl orphenyl; when there is a double bond between C2 and C3, R² is selectedthe group consisting of: (ia) C₅₋₁₀ aryl group, optionally substitutedby one or more substituents selected from the group comprising: halo,nitro, cyano, ether, carboxy, ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl andbis-oxy-C₁₋₃ alkylene; (ib) C₁₋₅ saturated aliphatic alkyl; (ic) C₃₋₆saturated cycloalkyl;

wherein each of R²¹, R²² and R²³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R² group is no more than 5;

wherein one of R²⁵a and R^(25b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy pyridyl and thiophenyl; and

where R²⁴ is selected from: H, C₁₋₃ saturated alkyl C₂₋₃ alkenyl, C₂₋₃alkynyl cyclopropyl phenyl, which phenyl is optionally substituted by agroup selected from halo, methyl, methoxy pyridyl and thiophenyl; whenthere is a single bond between C2 and C3, R² is

where R^(26a) and R^(26b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R²⁶a and R^(26b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester; when there is a doublebond between C2′ and C3′, R¹² is selected the group consisting of: (iia)C₅₋₁₀ aryl group, optionally substituted by one or more substituentsselected from the group comprising: halo, nitro, cyano, ether, carboxy,ester, C₁₋₇ alkyl, C₃₋₇ heterocyclyl and bis-oxy-C₁₋₃ alkylene; (iib)C₁₋₅ saturated aliphatic alkyl; (iic) C₃₋₆ saturated cycloalkyl;

wherein each of R³¹, R³² and R³³ are independently selected from H, C₁₋₃saturated alkyl, C₂₋₃ alkenyl, C₂₋₃ alkynyl and cyclopropyl, where thetotal number of carbon atoms in the R¹² group is no more than 5;

wherein one of R^(35a) and R^(35b) is H and the other is selected from:phenyl, which phenyl is optionally substituted by a group selected fromhalo, methyl, methoxy pyridyl, and thiophenyl; and

wherein R³⁴ is selected from: H, C₁₋₃ saturated alkyl, C₂₋₃ alkenyl,C₂₋₃ alkynyl, cyclopropyl, phenyl, which phenyl is optionallysubstituted by a group selected from halo, methyl, methoxy, pyridyl, andthiophenyl; when there is a single bond between C2′ and C3′, R¹² is

where R^(36a) and R^(36b) are independently selected from H, F, C₁₋₄saturated alkyl, C₂₋₃ alkenyl, which alkyl and alkenyl groups areoptionally substituted by a group selected from C₁₋₄ alkyl amido andC₁₋₄ alkyl ester; or, when one of R^(36a) and R^(36b) is H, the other isselected from nitrile and a C₁₋₄ alkyl ester; and p is from 1 to
 8. 2.The conjugate according to claim 1, wherein X is —CH₂—.
 3. The conjugateaccording to claim 1, wherein n is 1 to
 4. 4. The conjugate according toclaim 3, wherein n is
 2. 5. The conjugate according to claim 1, whereinthere is a double bond between C2 and C3, and R² is a C₁₋₅ saturatedaliphatic alkyl group.
 6. The conjugate according to claim 5, wherein R²is methyl, ethyl or propyl.
 7. The conjugate according to claim 1,wherein there is a double bond between C2 and C3, and R² is: (a) phenyl,which bears one to three substituent groups, wherein the substituentsmay be selected from methoxy, ethoxy, fluoro, chloro, cyano,bis-oxy-methylene, methyl-piperazinyl, morpholino and methyl-thiophenyl;(b) cyclopropyl; (c) a group of formula:

wherein the total number of carbon atoms in the R² group is no more than3; (d) the group:

or (e) a group of formula:

wherein R²⁴ is selected from H and methyl.
 8. The conjugate according toclaim 1, wherein there is a single bond between C2 and C3, R² is

and (a) R^(26a) and R^(26b) are both H; (b) R^(26a) and R^(26b) are bothmethyl; or (c) one of R^(26a) and R^(26b) is H, and the other isselected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl andalkenyl groups are optionally substituted.
 9. The conjugate according toclaim 1, wherein there is a double bond between C2′ and C3′, and R¹² isa C₁₋₅ saturated aliphatic alkyl group.
 10. The conjugate according toclaim 9, wherein R¹² is methyl, ethyl or propyl.
 11. The conjugateaccording to claim 1, wherein there is a double bond between C2′ andC3′, and R¹² is: (a) phenyl, which bears one to three substituentgroups, wherein the substituents are selected from methoxy, ethoxy,fluoro, chloro, cyano, bis-oxy-methylene, methyl-piperazinyl, morpholinoand methyl-thiophenyl; (b) cyclopropyl; (c) a group of formula:

wherein the total number of carbon atoms in the R¹² group is no morethan 3; (d) the group:

or (e) a group of formula:

wherein R³⁴ is selected from H and methyl.
 12. The conjugate accordingto claim 1, wherein there is a single bond between C2′ and C3′, R¹²is

and (a) R^(36a) and R^(36b) are both H; (b) R^(36a) and R^(36b) are bothmethyl; or (c) one of R^(36a) and R^(36b) is H, and the other isselected from C₁₋₄ saturated alkyl, C₂₋₃ alkenyl, which alkyl andalkenyl groups are optionally substituted.
 13. The conjugate accordingto claim 1, wherein DL is:


14. The conjugate according to claim 1, wherein the antibody comprises aVH domain having the sequence of SEQ ID NO.1.
 15. The conjugateaccording to claim 1, wherein the antibody comprises a VL domain havingthe sequence of SEQ ID NO.
 2. 16. The conjugate according to claim 1,wherein the antibody comprises a heavy chain having the sequence of SEQID NO. 3, or a heavy chain having the sequence of SEQ ID NO.
 11. 17. Theconjugate according to claim 1, wherein the antibody comprises a lightchain having the sequence of SEQ ID NO.
 4. 18. A composition comprisinga mixture of conjugates as defined in claim 1, wherein the average drugloading per antibody in the mixture of conjugates is about 1 to about 4.19. A pharmaceutical composition comprising the conjugate of claim 1,and a pharmaceutically acceptable diluent, carrier or excipient.
 20. Thepharmaceutical composition of claim 19, further comprising atherapeutically effective amount of a chemotherapeutic agent.
 21. Aconjugate of formula (I):Ab-(DL)_(p)  (I) wherein: Ab is an antibody that binds to DLK1 andcomprises a VH domain comprising a VH CDR1 with the amino acid sequenceof SEQ ID NO.5, a VH CDR2 with the amino acid sequence of SEQ ID NO.6,and a VH CDR3 with the amino acid sequence of SEQ ID NO.7; and, a VLdomain comprising a VL CDR1 with the amino acid sequence of SEQ ID NO.8,a VL CDR2 with the amino acid sequence of SEQ ID NO.9, and a VL CDR3with the amino acid sequence of SEQ ID NO.10; wherein DL is

and p is from 1 to
 8. 22. The conjugate according to claim 21, whereinthe antibody comprises a VH domain having the sequence of SEQ ID NO.1and a VL domain having the sequence of SEQ ID NO.
 2. 23. A compositioncomprising a mixture of conjugates as defined in claim 21, wherein theaverage drug loading per antibody in the mixture of conjugates is about1 to about
 4. 24. A pharmaceutical composition comprising the conjugateof claim 21, and a pharmaceutically acceptable diluent, carrier orexcipient.
 25. The pharmaceutical composition of claim 24, furthercomprising a therapeutically effective amount of a chemotherapeuticagent.